Methods and compositions for controlled polypeptide synthesis

ABSTRACT

Methods and compositions for the generation of polypeptides having varied material properties are disclosed herein. Methods include means for initiating the polymerization of aminoacid-N-carboxyanhydride (NCA) monomer by combining the monomer with an amido-containing metallacycle, for making self assembling amphiphilic block copolypeptides and related protocols for adding oligo(ethyleneglycol) functionalized aminoacid-N-carboxyanhydrides (NCAs) to polyaminoacid chains. Additional methods include means of adding an end group to the carboxy terminus of a polyaminoacid chain by reacting an alloc-protected amino acid amide with a transition metal-donor ligand complex to forming an amido-amidate metallacycle for use in further polymerization reactions. Novel compositions for use in peptide synthesis and design including five and six membered amido-containing metallacycles and block copolypeptides are also disclosed.

CROSS-REFERENCE-TO RELATED APPLICATIONS

This application is a continuation-in part of application Ser. No.09/568,121, which is a continuation-in part of non-provisionalapplication Ser. No. 09/272,109, filed Mar. 19, 1999, which claimedpriority under Section 119(e) to provisional application No. 60/078,649,filed Mar. 19, 1998. The 09/568,121 application also claims priorityunder Section 119(e) to provisional application Nos. 60/133,304, filedMay 10, 1999, 60/133,305 also filed May 10, 1999, 60/187,448 filed Mar.7, 2000, and 60/193,054 filed Mar. 29, 2000. In addition, the presentapplication claims priority under Section 119(e) to provisionalapplication No. 60/210,871 filed Jun. 8, 2000. The contents of theforegoing provisional and non-provisional applications are herebyincorporated by reference.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with Government support under Grant Nos. DMR9632716, CHE 9701969, and 9701969 awarded by the National ScienceFoundation, Grant No. N00014-96-0729 awarded by the Office of NavalResearch, and Grant No. DAAH04-96-1-004 awarded by the Department ofDefense. The Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the synthesis of amino-acid basedpolymers. In particular, this invention relates to methods andcompositions for the synthesis of amino-acid based polymers usingcatalysts under “living” conditions, that is conditions free oftermination and chain transfer.

2. Description of Related Art

Synthetic polypeptides have a number of advantages over peptidesproduced in biological systems and have been used to make fundamentalcontributions to both the physical chemistry of macromolecules and theanalysis of protein structures. See e.g. G. D. Fasman, Poly a-AminoAcids, Dekker, New York, (1967). Moreover, synthetic peptides are bothmore cost efficient and can possess a greater range of materialproperties than peptides produced in biological systems.

Small synthetic peptide sequences, typically less than 100 residues inlength, are conventionally prepared using stepwise solid-phasesynthesis. Such solid phase synthesis makes use of an insoluble resinsupport for a growing oligomer. A sequence of subunits, destined tocomprise a desired polymer, are reacted together in sequence on thesupport. A terminal amino acid is attached to the solid support in aninitial reaction, either directly or through a keying agent. Theterminal residue is reacted, in sequence, with a series of furtherresidues such as amino acids or blocked amino acid moieties to yield agrowing oligomer attached to the solid support through the terminalresidue. At each stage in the synthetic scheme, unreacted reactantmaterials are washed out or otherwise removed from contact with thesolid phase. The cycle is continued with a pre-selected sequence ofresidues until the desired polymer has been completely synthesized, butremains attached to the solid support. The polymer is then cleaved fromthe solid support and purified for use. The foregoing general syntheticscheme was developed by R. B. Merrifield for use in the preparation ofcertain peptides. See e.g. See Merrifield's Nobel Prize Lecture “SolidPhase Synthesis”, Science, Volume 232, pp. 341-347 (1986).

A major disadvantage of conventional solid phase synthetic methods forthe preparation of oligomeric materials results from the fact that thereactions involved in the scheme are imperfect; no reaction proceeds to100% completion. As each new subunit is added to the growing oligomericchain a small, but measurable, proportion of the desired reaction failsto take place. The result of this is a series of peptides, nucleotides,or other oligomers having deletions in their sequence. The result of theforegoing imperfection in the synthetic scheme is that as desired chainlength increases, the effective yield of desired product decreasesdrastically, since increased chances for deletion occur. Similarconsiderations attend other types of unwanted reactions, such as thoseresulting from imperfect blocking, side reactions, and the like. Ofequal, if not greater, significance, is the fact that the increasingnumbers of undesired polymeric species which result from the failedindividual reactions produce grave difficulties in purification. Forexample, if a polypeptide is desired having 100 amino acid residues,there may be as many as 99 separate peptides having one deleted aminoacid residue and an even greater possible number of undesired polymershaving two or more deleted residues, side reaction products and thelike.

Due to the above-mentioned problems associated with solid phasemethodologies, practitioners employ other protocols for peptidesynthesis. For example, synthetic copolymers of narrow molecular weightdistribution, controlled molecular-weight, and with block and stararchitectures can be prepared using so called living polymerizationtechniques. See e.g. O. Webster, Science, 251:887-893 (1991). In thesepolymerizations, chains grow linearly by consecutive addition ofmonomers, and chain-breaking transfer and termination reactions areabsent. The active end-groups of growing polymer chains do notdeactivate (i.e. they remain “living”) and chains continue to grow aslong as monomer is present. Chain length in living polymerizations iscontrolled through adjustment of monomer to initiator stoichiometry.Under circumstances when all chains grow at the same rate, livingpolymers will possess a narrow distribution of chain lengths. Complexsequences, such as block copolymers, are then built up by stepwiseaddition of different monomers to the growing chains. A. Noshay, et al.,Block Copolymers, Academic Press, New York, (1977).

The chemical synthesis of high molecular weight polypeptides is mostdirectly accomplished by the ring-opening polymerization ofα-aminoacid-N-carboxyanhydride (NCA) monomers (see equation 1 below).See e.g. H. R. Kricheldorf, in Models of Biopolymers by Ring-OpeningPolymerzation, Penczek, S. Ed., CRC Press, Boca Raton, (1990). Ingeneral terms, NCA polymerizations can be classified into two categoriesbased on the type of initiator used: either a nucleophile (typically aprimary amine) or strong base (typically a sodium alkoxide) (seeequation 1 below). Nucleophile initiated polymerizations are believed topropagate through a primary amine end-group (see equation 2 below).These polymerizations display complicated kinetics where an initial slowfirst order process is followed by accelerated monomer consumption:indicative of multiple propagating species with different reactivities.See e.g. M. Idelson, et al., J. Am. Chem. Soc., 80:2387-2393 (1958). Theprevalence of side reactions limit these initiators to the formation oflow molecular weight polymers (10 kDa<M_(n)<50 kDa) which typicallycontain a substantial fraction of molecules with degree ofpolymerization less than 10. As such, the polymers have very broadmolecular weight distributions (M_(w)/M_(n)=4-10). See e.g. R. D.Lundberg, et al., J. Am. Chem. Soc., 79:3961-3972 (1957).

Strong base initiated NCA polymerizations are much faster than amineinitiated reactions. These polymerizations are poorly understood but arebelieved to propagate through either NCA anion or carbamate reactivespecies (see equations 3 and 4 below, respectively). See e.g. C. H.Bamford, et al., Synthetic Polypeptides, Academic Press, New York,(1956).

A significant limitation of NCA polymerizations employing conventionalinitiators is due to the fact that they are plagued by chain-breakingtransfer and termination reactions which prevent formation of blockcopolymers. See e.g. H. R. Kricheldorf, a-Aminoacid-N-Carboxyanhydridesand Related Materials, Springer-Verlag, New York, (1987). Consequently,the mechanisms of NCA polymerization have been under intensive study sothat problematic side reactions could be eliminated. See e.g. H. R.Kricheldorf, in Models of Biopolymers by Ring-Opening Polymerization,Penczek, S. Ed., CRC Press, Boca Raton, (1990). These investigationshave been severely hindered by the complexity of the polymerizations,which can proceed through multiple pathways. Moreover, the highsensitivity of NCA polymerizations to reaction conditions and impuritieshas also led to contradictory data in the literature resulting incontroversy over the different hypothetical mechanisms. H. Sekiguchi,Pure and Appl. Chem., 53:1689-1714 (1981); H. Sekiguchi, et al., J.Poly. Sci. Symp., 52:157-171 (1975).

The significant problems with existing peptide synthesis methodologiescreate a variety of problems for practitioners. For example, the chainbreaking transfer reactions that occur in the NCA polymerizationspreclude the systematic control of peptide molecular weight. Moreover,block copolymers cannot be prepared using such methods.

Block copolymers of amino acids have been less well studied, largelybecause our synthetic methods do not yet have fine enough control toproduce well-defined structures. F. Cardinauz, et al., Biopolymers,16:2005-2028 (1977). The same is true of the synthesis of blockcopolypeptides for use as biomaterials or as selective membranes—thepotential advantages of the protein-like architectures have remainedunrealized for want of adequate synthetic building blocks and tools.

For example, biomedical applications, such as drug delivery typicallyrequire water-soluble components to enhance their ability forcirculation in vivo. The problem with common water-soluble polypeptides(e.g., poly-L-lysine and poly-L-aspartate) is that they arepolyelectrolytes that display pH-dependent solubility and limitedcirculation lifetime due to aggregation with oppositely chargedbiopolymers. Nonionic, water-soluble polypeptides are desired forbiomedical applications since they avoid these problems, and can alsodisplay the stable secondary structures of proteins that influencebiological properties. However, all high molecular weight nonionichomopolypeptides (>25 residues) derived from naturally occurring aminoacids are notoriously insoluble in water.

One approach to producing nonionic water-soluble polypeptides employspolyethylene glycol (PEG), which is typically grafted onto polypeptidesor other polymers to improve their properties in vivo. PEG is nonionic,water-soluble, and most importantly not recognized by immune systems. Itis believed that PEG imparts biocompatibility through formation of ahydrated “steric barrier” at the surface of material that cannot bepenetrated or recognized by biological molecules, such as proteolyticenzymes. As such, block or graft copolymer drug carriers containing PEGare able to circulate for long periods in the bloodstream withoutdegradation.

Despite its attractive properties, a drawback to grafting PEG ontopolypeptides is the need for expensive amino- orcarboxylato—functionalized molecules for coupling, which typically mustbe short (<5,000 Da) to ensure high functionalization. Accordingly,there remains considerable interest in developing alternative methodsfor producing nonionic water-soluble polypeptide building blocks thatalso incorporate the attractive properties of biochemical stability,self-assembly and water solubility into polypeptides.

Polypeptides are being considered for a variety of biomedical problemssuch as tissue engineering and drug delivery. Another consideration forthese applications is the incorporation of end group functionality ontothe chains, which is essential for targeting of the drug deliverycomplexes as well as substrate specific anchoring of these materials.These, and other features would be useful for controlling both thestructure and the properties of polypeptide materials. Consequently,there is a need for novel methods and compositions which allow for thefacile generation of peptides tailored to have specific desirableproperties.

SUMMARY OF THE INVENTION

The present invention discloses novel methods and compositions whichaddress the need for advanced tools to generate polypeptides havingvaried material properties. The methods and initiator compositions forNCA polymerization disclosed herein allow the precise control of suchpolypeptide synthesis. In particular, the methods of the invention allowsuccessful peptide synthesis by utilizing the versatile chemistry oftransition metals to mediate the addition of monomers to the activepolymer chain-ends, and therefore eliminate chain-breaking sidereactions in favor of the chain-growth process. In this way, thedisclosed methods allow the formation of block copolymers. Moreover, bybinding the active end-group of the growing polymer to a metal center,its reactivity toward monomers can be precisely controlled throughvariation of the metal and ancillary ligands bound to the metal. Thewide range of selective chemical transformations and polymerizationswhich are catalyzed by transition metal complexes attests to theversatility of this approach.

One embodiment of the invention provides a method of making anamido-containing metallacycle comprising combining an amount of ana-aminoacid-N-carboxyanhydride monomer with an initiator moleculecomprising a low valent transition metal-Lewis Base ligand complex sothat an amido-containing metallacycle is formed.

An alternative embodiment of the invention provides a method of makingan initiator molecule, which includes the step of combining anallyloxycarbonyl (alloc) protected amino acid amide and a low valenttransition metal-Lewis base ligand complex so that an amido-amidatemetallacycle is formed having the following general formula:

wherein M is a low valent transition metal, L is a Lewis base ligand;one of R1 and R2 is an amino acid side group and the other is hydrogen;and R3 is any functional end group capable of being attached to aprimary amine group. The R3 end group will typically be used to “tag” orfunctionalize the polypeptide chains, and is the main advantageassociated with using this method. Typically, this group will be apeptide, oligosaccharide, oligonucleotide, fluorescent molecule, polymerchain, small molecule therapeutic, chemical linker to attach thepolypeptide to a substrate, chemical linker to act as a sensing moiety,or reactive linker to couple the polypeptide to larger molecules such asproteins, polysaccharides or polynucleotides

Another embodiment of the invention provides compositions consisting offive or six membered amido-containing metallacycles comprising moleculesof the general formula:

wherein M is a low valent transition metal;

L is a Lewis Base ligand;

each of R1, R2, R3, R5 and R6 (independently) is a moiety selected fromthe group consisting of the side chains of alanine, arginine,asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, threonine, tryptophan, tyrosine or valine; R4 is ahydrogen moiety or a polyaminoacid chain; and R7 is a functional endgroup.

In preferred embodiments of these compositions, the metal is atransition metal selected from the group consisting of nickel,palladium, platinum, cobalt, rhodium, iridium and iron and the LewisBase ligand is selected from the group consisting of pyridyl ligands,diimine ligands, bisoxazoline ligands, alkyl phosphine ligands, arylphosphine ligands, tertiary amine ligands, isocyanide ligands andcyanide ligands.

A related embodiment is method of initiating an a-amino acid—N-carboxyanhydride monomer polymerization by combining an NCA monomerwith an initiator molecule comprising an amido-containing metallacycle,which contains a nucleophilic alkyl amido group stabilized by a rigidchelate and a non-nucleophilic proton-accepting group. In preferredversions, the proton-accepting group is selected from the group of amidosulfonamidate, an amido-amidate having an extracyclic nitrogen, anamido-ureate, and amido-carbamate, or an amido-aldimate.

A related embodiment of the invention consists of a method of adding anaminoacid-N-carboxyanhydride (NCA) to a polyaminoacid chain having anamido containing metallacycle end group by combining the NCA with thepolyaminoacid chain so that the NCA is added to the polyaminoacid chain.

Another embodiment of the invention disclosed herein entails a method ofpolymerizing aminoacid-N-carboxyanhydride monomers by combining a NCAmonomer with an initiator molecule complex comprised of a low valenttransition metal-Lewis Base ligand. A specific embodiment of theinvention disclosed herein entails a method of polymerizingaminoacid-N-carboxyanhydride monomers having a ring with a O—C₅ and aO—C₂ anhydride bond which consists of combining a first NCA monomer withan initiator molecule complex comprised of a low valent metal capable ofundergoing an oxidative addition reaction wherein the oxidative additionreaction formally increases the oxidation state by two electrons; and anelectron donor comprising a Lewis base. The initiator molecule is thenallowed to open the ring of the first NCA through oxidative additionacross either the O—C₅ or O—C₂ anhydride bond and then combine with asecond NCA monomer, to form an amido-containing metallacycle. A thirdNCA monomer is then allowed to combine with the amido containingmetallacyle so that the amido nitrogen of the amido containingmetallacyle attacks the carbonyl carbon of the NCA. Thus, the NCA isadded to the polyaminoacid chain and the amido containing metallacyle isregenerated for further polymerization. In a preferred embodiment of theinvention, the efficiency of the initiator is controlled by allowing thereaction to proceed in a solvent selected for its ability to influencethe reaction. In a specific embodiment of the invention, the solvent isselected from the group consisting of ethyl acetate, toluene, dioxane,acetonitrile, THF and DMF.

Another embodiment of the invention provides a method of making a blockcopolypeptide consisting of combining an amount of a firstaminoacid-N-carboxyanhydride (NCA) monomer with an initiator moleculecomprising a low valent transition metal-Lewis Base ligand complex sothat a polyaminoacid chain is generated and then combining an amount ofa second aminoacid-N-carboxyanhydride monomer with the polyaminoacidchain so that the second aminoacid-N-carboxyanhydride monomer is addedto the polyaminoacid chain. In a preferred embodiment of this method,the initiator molecule combines with the firstaminoacid-N-carboxyanhydride monomer to form an amido containingmetallacycle intermediate of the general formula:

wherein M is the low valent transition metal;

L is the Lewis Base ligand;

each of R1, R2 and R3 independently is a moiety selected from the groupconsisting of the side chains of alanine, arginine, asparagine, asparticacid, cysteine, glutamic acid, glutamine, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine or valine; and

R4 is the polyaminoacid chain.

In yet another embodiment, the invention provides block copolypeptidecompositions having characteristics which have been previouslyunattainable through conventional techniques. A specific embodiment ofthis invention consists of a polypeptide composition comprising a blockpolypeptide having a number of overall monomer units that are greaterthan about 100 amino acid residues and a distribution of chain-lengthsat least about 1.01<Mw/Mn<1.25. In a related embodiment, the polypeptidehas a number of overall monomer units that are greater than about 250amino acid residues. In a specific embodiment, the copolypeptideconsists of a least 3 blocks of consecutive identical amino acid monomerunits. In a specific embodiment of this invention, at least one of theblock's components is g-benzyl-L-glutamate.

The present invention also discloses novel methods and compositions,which address the need for biocompatible materials having improvedproperties of biochemical stability, water solubility, and selfassembly. The methods of making amphiphilic block copolypeptidesdisclosed herein allow the synthesis and assembly of compositionscontaining well-defined vesicular structures, which are potentiallyvaluable for biomedical applications, such as drug delivery.

One embodiment of the invention provides a method of making anamphiphilic block copolypeptide, which includes the steps of (1)generating a soluble block polypeptide by combining an amount of anoligo (ethyleneglycol) functionalized aminoacid-N-carboxyanhydride(EG-aa-NCA) monomer with an initiator molecule; and (2) attaching aninsoluble block by combining the soluble block with a compositioncomprising at least one other amino acid NCA monomer. In preferredembodiments of this method, the amino acid component of the EG-aa-NCAmonomer is lysine, serine, cysteine, or tyrosine, whereas the insolubleblock can contain a mixture of amino acids, which includes one or morenaturally occurring amino acids.

A related embodiment of the invention consists of a method of adding anaminoacid-N-carboxyanhydride (NCA) to a soluble block polypeptide havingone or more oligo(ethyleneglycol)-terminated amino acid residues bycombining the NCA with the polypeptide so that the NCA is added to thepolypeptide.

In yet another embodiment, the invention provides amphiphilic blockcopolypeptide compositions, which have improved characteristics ofsolubility, biochemical stability and biocompatibility. The amphiphilicblock copolypeptide includes a soluble block polypeptide having one ormore oligo(ethyleneglycol)-terminated amino acid residues and aninsoluble block comprised substantially of nonionic amino acid residues.A specific embodiment of this invention is a polypeptide compositioncomprising: (1) a soluble block polypeptide having EG-lysine residues,and (2) an insoluble block polypeptide containing a mixture of two tothree different kinds of amino acid components in a statistically randomsequence. In another specific embodiment, the copolypeptide consists ofa least 3 blocks, wherein one or more of the blocks is a soluble blockpolypeptide and another block is an insoluble block polypeptide.

The amphiphilic nature of the block copolypeptides provides yet anotherembodiment, which is a method of forming vesicles. This method consistsof suspending the amphiphilic block copolypeptides in an aqueoussolution so that the copolypeptides spontaneously self assemble intovesicles. In a specific embodiment, smaller vesicles having a diameterof about 50 nm to about 500 nm can be formed by sonicating thesuspension of larger vesicles.

In a related embodiment, the invention provides vesicle-containingcompositions comprised of the amphiphilic block copolypeptides of thepresent invention and water.

In another related embodiment, the invention provides methods for makingEG-functionalized amino acid monomers, which includes the step ofcombining an ethyleneglycol (EG) derivative with an amino acid having areactive side group, e.g., lysine, serine, cysteine, and tyrosine.

The methods and compositions for making amphiphilic block copolypeptidesare particularly attractive since the EG-amino acid domains will emulatecertain desirable features of poly (ethyleneglycol), PEG. For example,PEG is well known for its bioinvisibility meaning that it is notrecognized by immunological defense mechanisms in the body, and thus hasfound many useful applications in drug delivery, enzyme stabilization,tissue engineering, and implant surface modification.

As examples of preferred embodiments of the invention, a series ofinitiators for the polymerization of amino acid-N-carboxyanhydrides(NCAs) into block copolypeptides based on a variety of metals andligands are described. These initiators are substantially different innature from all known conventional initiators used to polymerize NCAsand are also unique in being able to control these polymerizations sothat block copolymers of amino acids can be prepared. Specifically,these initiators eliminate chain transfer and chain termination sidereactions from these polymerizations resulting in narrow molecularweight distributions, molecular weight control, and the ability toprepare copolymers of defined block sequence and composition. All ofthese traits have previously been unobtainable using conventionalinitiator systems. Furthermore, the initiators described herein arereadily prepared in a single step from commercially available materials.

The discovery of this new class of initiators and methods for their useallows for the elimination of side reactions from NCA polymerizationsand further allows the preparation of well-defined block copolypeptides.Formation of an illustrative example of our initiator results from theoxidative-addition reaction of an NCA monomer to a zerovalent nickelcomplex, bipyNi(COD); bipy 2,2′-bipyridyl, COD=1,5-cyclooctadiene. Thisreaction is similar to the known oxidative-addition of cyclic anhydridesto zerovalent nickel to yield acyl-carboxylato divalent nickel complexes(see equation 5 below).

While this reaction is similar to these known oxidative-additionreactions, the reaction occurring in the formation of the moleculesdisclosed herein is without precedent.

The methods and initiator compositions disclosed herein allow thepreparation of complex polypeptide biomaterials which have potentialapplications in biology, chemistry, physics, and materials engineering.Potential applications include medicine (drug delivery, tissueengineering), “smart” hydrogels (environmentally responsive organicmaterials), and in organic/inorganic biomimetic composites (artificialbone, high performance coatings).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 compares the abilities of different initiators to controlmolecular weight of PBLG as a function of initiator concentration inpolymerizations of Glu-NCA: A, phenethylamine initiator; B, bipyNi(COD)initiator; C, sodium tert-butoxide initiator; D, theoretical molecularweight calculated from (M)₀/[I]₀. All polymerizations were run inanhydrous DMF at 25° C. for 1 day in sealed tubes. Molecular weight(M_(n)) was determined by tandem GPC/light scattering in 0.1M LiBr inDMF at 60° C.

FIG. 2 is a chromatogram of a PBLG_(0.78)-b-PZLL_(0.22) diblockcopolymer prepared by sequential addition of Lys-NCA and Glu-NCA tobipyNi(COD) initiator in DMF. The polymer was injected directly into theGPC, eluted using 0.1M LiBr in DMF at 60° C. through 10⁵ Å and 10 ³ ÅPhenomenex 5 μm columns, and detected with a Wyatt DAWN DSP lightscattering detector and Wyatt Optilab DSP.

FIG. 3 shows 2 chemical reaction schemes associated with amino acidderived nickelacycles, intermediates in nickel initiator mediatedpolypeptide synthesis.

FIG. 4 shows 4 chemical reaction equations associated with amino acidderived nickelacycles, intermediates in nickel initiator mediatedpolypeptide synthesis.

FIG. 5 shows chemical structures of some ligands used in NCApolymerization reactions

FIG. 6 shows the formation of an amido-containing metallacycle byreaction of NCAs with a metal initiator.

FIG. 7 shows MALDI-MS of leucyl isoamylamide-C-terminatedoligo(phenylglycine)s. A partial expansion of the spectrum is shown inthe upper right. Mass series were observed for (a) leucine-OH terminatedoligomers resulting from the hydrolysis of the terminal amide in TFA,(b) Leucine isoamylamide terminated oligomers resulting from intactend-functionalized chains, and (c) non-functionalized chains. Forexample, 9a indicates the MH+ ion of the nona(phenylglycine) of the aseries, the b and c series are labeled similarly. The ions of thevarious series also formed adducts with O atoms and CO₂, and are labeledas such.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The term “block copolypeptide” as used herein refers to polypeptidescontaining at least two covalently linked domains (“blocks”), one blockhaving amino acid residues that differ in composition from thecomposition of amino acid residues of another block. The number, length,order, and composition of these blocks can vary to include all possibleamino acids in any number of repeats. Preferably the total number ofoverall monomer units (residues) in the block copolypeptide is greaterthan 100 and the distribution of chain-lengths in the block copolymer isabout 1.01<Mw/Mn<1.25, where Mw/Mn=weight average molecular weightdivided by number average molecular weight.

The terms “protection” and “side-chain protecting group” as used hereinrefer to chemical substituents placed on reactive functional groups,typically nucleophiles or sources of protons, to render them unreactiveas protic sources or nucleophiles. The choice and placement of thesesubstituents was according to literature procedures. M. Bodanszky, etal., The practice of Peptide Synthesis, 2^(nd) Pd Ed., Springer,Berlin/Heidelberg, (1994).

II. Overview

The ideal living polymerization is characterized by fast initiation andan absence of the termination and chain transfer steps that in mostpolymerization systems compete with propagation of the growing chain.When these conditions are realized, all polymer chains begin growing atabout the same time and continue to grow until the monomer has beenexhausted. The average number of monomer residues per chain is thensimply the molar ratio of monomer to initiator, and the distribution ofchain lengths is described by Poisson statistics. M. Szwarc, Carbanions,Living Polymers, and Electron-Transfer Processes, Wiley, New York(1968). As disclosed herein, these conditions have been met in thepolymerization of NCAs by bipyNi (COD). The distribution of chainlengths is narrow, consistent with Poission statistics, and the rate ofpolymerization is proportional to monomer concentration, indicating thatthe number of active chain ends remains constant throughout thereaction.

It is the absence of termination and transfer that makes livingpolymerization so powerful for synthesizing block copolymers. Becausethe growing chains remain active even after the monomer has beenexhausted, adding a second monomer at that stage results in the growthof a second block distinct in composition from the first. Proper choiceof monomers allows one to engineer the kinds of combinations ofproperties described above: rubbery glassy; hydrophilic and hydrophobic;conducting and insulating; and so on.

By providing examples of NCAs polymerized by zero-valent nickelcatalysts under ‘living’ polymerization conditions; that is, conditionsfree of termination and chain transfer, the disclosed methods andcompositions allow for the generation and manipulation of peptides inmanners that have not previously been possible. Living polymerizationsallow the synthesis of polymers of predetermined molecular weights andnarrow molecular-weight distribution; and, perhaps more importantly, thepreparation of well-defined block copolymers in which long sequences ofeach of the individual monomer residues are linked together at a singlesite. The advantages of living polymerizations, which once were reservedfor a small subset of polymerizable monomers, can now be extended toNCAs and to the preparation of high-molecular-weight polypeptides andblock copolypeptides with unusual and useful properties.

The methods and compositions disclosed herein teach new ways topolymerize amino acids and to add amino acids to polyamino acid chains.Further, the initiators and amido-containing metallacyle compositionsdisclosed herein allow the synthesis of block copolypeptides byeliminating of side reactions in favor of the chain-growth process (i.e.living polymerization), thus allowing multiple monomer additions topolyaminoacid chains. While the specific methods and initiator andamido-containing metallacyle compositions disclosed represent preferredembodiments of this invention, as discussed below, other embodiments arealso contemplated.

In the examples below, general features for the formation of activemetal initiators are discussed as well a means to determine initiatorefficiency (see e.g. Example 3). Moreover disclosed herein areparameters for generating effective initiators as well assays to assessthe activity of different initiator complexes and their ability tofunction in the disclosed methods. Moreover, disclosed herein are anumber of different initiator compositions which were evaluated fortheir ability to work in this system. In addition, the Examplesillustrate the effects of different solvents on the various polypeptideaddition reactions. Using these protocols, one skilled in the art mayconstruct and then assess the ability of a new potential initiatormolecule to function in the disclosed methods. Using the protocolsdisclosed herein, one may also assess the activity of differentamido-containing metallacycles and their ability to function in thedisclosed methods.

In providing new means to polymerize amino acids and to add amino acidsto polyamino acid chains, the disclosed methods and compositionsovercome a number of problems associated with complex polypeptidesynthesis. Successful block copolypeptide synthesis requires eliminationof side reactions in favor of the chain-growth process (i.e. livingpolymerization), thus allowing multiple monomer additions to each chain.L. J. Fetters, “Monodisperse Polymers” in Encyclopedia of PolymerScience and Engineering 2nd Ed., Wiley-Interscience, New York, 10:19-25(1987); O. Webster, “Living Polymerization Methods” Science, 251:887-893(1991). This problem was addressed by utilizing the versatile chemistryof transition metals to mediate the addition of monomers to the activepolymer chain-ends. T. J. Deming, “Polypeptide Materials: New SyntheticMethods and Applications” Adv. Materials, 9:299-311 (1997). The widerange of selective chemical transformations and polymerizations that arecatalyzed by transition metal complexes attests to the potential of thisapproach. J. P. Collman, et al., Principles and Applications ofOrganotransition Metal Chemistry 2nd Ed., University Science, MillValley, (1987).

The oxidative addition of cyclic carboxylic acid anhydrides to nickel(0)was first reported by Uhlig and coworkers. E. Uhlig, et al., Z. Anorg.Allg. Chem., 465:141-146 (1980). When succinic anhydride is added toL₂Ni(COD) a six-membered acyl-carboxylato nickelacycle is initiallyformed which decarbonylates above ambient temperature to form a stablefive-membered alkyl-carboxylato complex. L₂=donor ligand(s);COD=1,5-cyclooctadiene; bipy=2,2′-bipyridyl. With unsymmetricanhydrides, the regioselectivity of oxidative addition was found to varywith the donor ligand (L₂) and solvent. A. M. Castano, et al.,Organometallics, 13:2262-2268 (1994). When an NCA oxidatively adds tonickel(0) across the unsymmetric anhydride linkage, regioselectivity ofaddition is important in determining the nature and reactivity of theproducts. With both initial products, decarbonylation would be expectedto be favored over decarboxylation due to the greater stability of theresulting five-membered metallacycles (see scheme 1 of FIG. 3). E.Uhlig, et al., Z. Anorg. Allg. Chem., 465:141-146 (1980). The additionof NCAs to nickel(0) is of interest because the resulting metal-amido ormetal-carbamato complexes might prove useful as reactive, chiralsynthetic intermediates.

The reaction chemistry of a-amino acid-N-carboxyanhydrides (NCAs) hasbeen under study since these molecules are potential precursors tosequence specific peptides, polypeptides, and other amino acidcontaining compounds. H. R. Kricheldorf, a-Aminoacid-N-Carboxyanhydridesand Related Materials, Springer-Verlag, New York, (1987); H. R.Kricheldorf, in Models of Biopolymers by Ring-Opening Polymerization,Penczek, S. Ed., CRC Press, Boca Raton, (1990). NCAs are attractivepeptide building blocks since they are readily prepared from amino acidsand since they show no racemization at the chiral a-carbon either duringpreparation or in subsequent reactions. W. E. Hanby, et al., Nature,161.132 (1948); A. Berger, et al., J. Am. Chem. Soc., 73:408-44088(1951). Utilization of NCAs, however, has been limited because of theircomplicated reactivity and tendency to uncontrollably polymerize.

Attempts to use metal coordination complexes of conventional amineinitiators to control the polymerizations have been described in theart. T. J. Deming, “Transition Metal-Amine Initiators for Preparation ofWell-Defined Poly(g-benzyl-L-glutamate)” J. Am. Chem. Soc., 1997,119:2759-2760 (1997). Use of metal-amine complexes for polymerization ofg-benzyl-L-glutamate N-carboxyanhydride, Glu-NCA as described herein,allowed the preparation of poly(g-benzyl-L-glutamate), PBLG, with narrowmolecular weight distribution (M_(w)/M_(n)=1.05-1.10) and some controlover molecular weight. However, typical problems inherent in primaryamine initiated polymerizations (i.e. slow propagation and chaintransfer reactions) prevented use of these initiators for preparation ofblock copolypeptides.

The living polymerization of NCAs and synthesis of block copolypeptidesusing nickel initiators has been reported. T. J. Deming, Nature,390:386-389 (1997). This reference discloses stoichiometric reactionswhere NCAs oxidatively add regioselectively to sources of zerovalentnickel to yield complexes which subsequently rearrange to unprecedentedamido-containing metallacycles. When complexed with donor ligands, thenickelacycles are efficient NCA polymerization initiators.

III. Methods and Compositions of the Invention

Unlike the initiators known in the art, the molecules described hereinare a new class of initiators based on low valent metal-Lewis basecomplexes which are able to eliminate significant competing terminationand transfer steps from NCA polymerizations and allow preparation ofwell-defined block copolypeptides.

Donor Ligand/Transition Metal Complexes

A variety of illustrative initiator complexes useful in the generationof block copolypeptides are described herein such as those generatedusing bis-1,5-cyclooctadiene nickel (Ni(COD)₂) as the nickel source and2,2′-bipyridyl (bipy) as the donor ligand component in tetrahydrofuran(THF) solvent. As discussed below and as shown in Tables 7 and 8, theuse of other sources of zerovalent nickel (e.g. nickel-olefin complexes,nickel-carbonyl complexes, nickel-isocyanide or cyanide complexes, andother specific ligands such as PR₃ [R=Me, Et, Bu, cyclohexyl, phenyl],R₂PCH₂CH₂PR₂ [R=Me, phenyl], a, a′-diimine ligands [1,10-phenanthroline,neocuproine], diamine ligands [tetramethylethylene diamine], andisocyanide ligands [tert-butyl isocyanide and related nickel nitrogen orphosphorous donor ligand complexes) can work in the complexes of thepresent invention to initiate these polymerizations.

As shown in Example 4 below, in addition to bis-1,5-cyclooctadienenickel (Ni(COD)₂), other sources of zerovalent nickel (e.g. Ni(CO)₄) aswell as other low valent metals in the initiator complexes have beenused successfully in these methods. Illustrative metals useful in thegeneration of initiators are “low valent” transition metals, inparticular the metals of Group VIII of the Periodic Table andillustrative examples of initiators using such metals is provided inTable 8. This group includes the metals, Fe, Ru, Os, Co, Rh, Ir, Ni, Pdand Pt. The “low valent” forms of the metals implies that the metals arein low-oxidation states. For Ni, Pd, Pt, Co, and Fe this means thezerovalent (O) oxidation state. For Ir and Rh, this means the monovalent(+1) oxidation state. For Ru and Os, this means the divalent (+2)oxidation state. See the complexes in Table 8 for relevant examples. Theterm, “Low valent metal” also extends to other metals in an oxidationstate such the metal may undergo a 2 electron oxidation.

As shown in Example 4 below, in addition to using 2,2′-bipyridyl (bipy)as the donor ligand component, a variety of other donor ligands can beused in the initiator complexes. As shown in Table 7, ligands which canbe used to bind to the initiator metals complexes need to comprise a nonnucleophilic electron donor comprising a Lewis base and can consists ofa variety of groups which have this property including those that arepyridyl based (see e.g. entry 2-144, Table 7), diimine (see e.g. DIPRIM,2-148), bisoxazoline (see e.g. DPOX, 3-2), alkyl phosphine (see e.g.dmpe, 2-148), aryl phosphine (see e.g. PPh₃, 2-151), tertiary amine (seee.g. tmeda, 3-10), isocyanide or cyanide (see e.g. 3-34), p-cymene orcombinations of these ligands. Generally, the ligands are bidentate(coordinate through 2 atoms) or are composed of 2 equivalents ofmonodentate ligands. Tridentate ligands can also be used (e.g.terpyridine). The ligands generally are bound to the metal through N, P,or C atoms of the molecule. Other N or P donor ligands, similar to thosementioned above (i.e. neutral, non-nucleophilic, aprotic) can alsosupport these initiators.

NCA Monomers

As illustrated in the references cited above, NCA monomers are wellknown in the art (see e.g. H. R. Kricheldorf,a-Aminoacid-N-Carboxyanhydrides and Related Materials, Springer-Verlag,New York, (1987)). Moreover, the use of a variety of NCA monomers inmethods of polypeptide synthesis is well known in the art. For examplethe stepwise synthesis of polypeptides using NCAs (or derivativesthereof) is disclosed in U.S. Pat. No. 3,846,399 (incorporated byreference herein). In addition, U.S. Pat. No. 4,267,344 disclosesN-substituted N-carboanhydrides of a amino acids and their applicationin the preparation of peptides (incorporated by reference herein).

Another embodiment of the present invention involves the synthesis ofunique oligo (ethyleneglycol) functionalized amino acids and theirsubsequent polymerization into oligo (ethyleneglycol) functionalizedpolypeptides. The method of making these monomers includes the step ofcombining an ethyleneglycol (EG) derivative with an amino acid having areactive side group, e.g., lysine, serine, cysteine, and tyrosine, toform an EG-functionalized amino acid. The EG derivative has the generalformula (CH₃OCH₂CH₂)_(n)X, where n amounts to about 1 to 3 EG repeatsand X is a reactive group, such as chloroformate, N-hydroxysuccidimydylacetate, or a halide. The EG functionalized amino acids can then beconverted to NCA monomers for use in the synthesis of oligo (EG)functionalized polypeptides.

A generalized scheme for synthesizing oligo(EG) functionalized serine,tyrosine and cysteine is shown in Scheme I (below).

More detailed synthetic routes are illustrated in Scheme II and inExample 5 (below).

In addition, a preferred method of synthesizing oligo(EG) functionalizedlysine is shown in Scheme III (below).

These amino acid derivatives and polymers are new compositions ofmatter, and the polymers possess unique properties which make thempotentially valuable for biomedical/biotechnological applications. Inpreferred embodiments polyaminoacid chains are synthesized having atleast 10 consecutive oligo-EG functionalized amino acid side chains.Moreover, oligo-EG functionalized NCA monomers can be used to makepolypeptide chains where the side chain of every residue is capped by aethylene glycol oligomer; in effect, “PEG coated polypeptides”.Poly(ethyleneglycol), PEG, is well known for its “bioinvisibility”,meaning that it is not recognized by immunological defense mechanisms inthe body (non-antigenic), and thus has found many useful applications indrug delivery, enzyme stabilization, tissue engineering, and implantsurface modification.

Such polymers are unusual in possessing excellent water solubility overbroad pH ranges (2-13) and salt concentrations. Furthermore, the PEG“coating” strongly stabilizes the secondary structure of thepolypeptides (beta-sheets and alpha-helices) such that the polymerspossess stable secondary structures over broad pH and temperatureranges. These new polymers are attractive since they display most of thesame properties of PEG (water solubility, biocompatibility), but possesscompletely different chain structures. The serine and cysteine-derivedpolymers adopt beta-sheet structures and represent the first examples ofwater soluble polypeptides that form stable beta-sheet structures. Assuch their solution and mechanical properties are markedly differentfrom PEG and thus provide an interesting alternative to PEG inbiomedical materials.

Making Amido-Containing Metallacycles

The initiator complexes of the present invention can be synthesized bytwo different approaches, both of which entail the use of a low valenttransition metal-Lewis Base ligand complex and result in the formationof an amido-containing metallacycle.

Transition Metal/Donor Ligand+NCA Monomer

One embodiment of the invention provides a method of making anamido-containing metallacycle comprising combining an amount of ana-aminoacid-N-carboxyanhydride monomer with an initiator moleculecomprising a low valent transition metal-Lewis Base ligand complex sothat an amido-containing metallacycle is formed. Formation of theseinitiators results from the unprecedented reaction of an NCA monomerwith a low valent metal-Lewis base complex such as a zerovalent nickelcomplex bipyNi(COD); bipy=2,2′-bipyridyl, COD=1,5-cyclooctadiene. Thisreaction is similar to the oxidative-addition of cyclic anhydrides tozerovalent nickel which yields divalent nickel metallacycles (seeequation 6 below). E. Uhlig, et al., □Reaktionen cyclischerCarbonsaeureanhydride mit (a,a′-Dipyridyl)-(cyclooctadien-1,5)-nickel□Anorg. Allg. Chem., 465:141-146 (1980); K. Sano, et al., “Preparation ofNi- or Pt-Containing Cyclic Esters by Oxidative Addition of CyclicCarboxylic Anhydrides and Their Properties” Bull. Chem. Soc. Jpn.,57:2741-2747 (1984); A. M. Castaño, et al., “Reactivity of aNickelacycle Derived from Aspartic Acid: Alkylations, Insertions, andOxidations” Organometallics, 13:2262-2268 (1994).

Activation and polymerization of NCAs through oxidative ring opening ofthe anhydride, however, is without precedent. Successful polymerizationof L-proline NCA, which lacks a proton bound to nitrogen, usingbipyNi(COD) supports the hypothesis of oxidative addition across theanhydride bond, rather than reaction at the N—H bond. In this context,it is observed that the initial oxidative addition to the NCA can occurat either side of the anhydride bond (for example, O—C₅ for nickel,cobalt and iron and both O—C₅ and O—C₂ for rhodium and iridium).

Since NCAs are unsymmetrical anhydrides, the oxidative-addition of NCAscan yield two distinct isomeric products In practicing one embodiment ofthe invention, it is found that the addition of NCAs to nickel wascompletely regioselective for ring opening across the O—C₅ bond.Reaction of bipyNi(COD) with ¹³C₂-L-leucine NCA and ¹³C₅-L-leucine NCAyielded oxidative addition products and bipyNi(CO)₂ which were examinedby ¹³C NMR and FTIR spectroscopy. Detection of bipyNi(¹²CO)₂ (FTIR(THF):n(CO)=1978, 1904 cm⁻¹) from the reaction of ¹³C₂-L-leucine NCA, andbipyNi(¹³CO)₂ (FTIR(THF): n(CO)=1934, 1862 cm⁻¹; ¹³C NMR (DMF-d₇): d 198(Ni—CO)) from the reaction of ¹³C₅-L-leucine NCA identified theregiochemistry of the product. In dimethylformamide (DMF), a goodsolvent for polypeptides, this addition product was found to becompletely active for polymerization of additional NCA monomers.

Donor Ligand/Transition Metal+Alloc-Amino Acid Amide

The amido-amidate metallacycles generated from low valent transitionmetal precursors, as described above, are active intermediates in thecontrolled polymerization of α-amino acid-N-carboxyanhydrides (NCAs). Alimitation of this methodology is that the active propagating speciesare generated in situ and thus do not allow for controlledfunctionalization of the polypeptide chain ends. For this reason, wepursued alternative methods for the direct synthesis of these types ofinitiators.

Thus, another embodiment of the present invention entails new tandemaddition reactions that allow the general synthesis of amido-amidatemetallacycles useful for preparation of polypeptides containing avariety of defined endgroups. This method of making an initiatormolecule includes the step of combining an allyloxycarbonyl (alloc)protected amino acid amide and a low valent transition metal-Lewis baseligand complex so that an amido-amidate metallacycle is formed havingthe following general formula:

wherein M is a low valent transition metal, L is a Lewis base ligand;one of R1 and R2 is an amino acid side group and the other is hydrogen;and R3 is any functional end group capable of being attached to aprimary amine group.

The alloc-amino acid amide has the general formulaAlloc-NH—CH(R′)C(O)NHR″ where R′ is an amino acid side group and R″ is afunctional end group. The “alloc” group includes at least one allylicmoiety, i.e., a carbon-carbon double bond bound to a saturated carbon.The backbone “allylic” system is a key element which is required for thereaction to work. The allylic backbone is, in turn, coupled via anoxygen to the carbonyl bound to the nitrogen of the amino acid. The R′group can be a side-chain functionality found for any of the 20 naturalamino acids (L-form), their corresponding D-forms, or unnaturalsynthetic amino acid side-chains, providing that reactive functionalgroups (those that are either protic or nucleophilic—such as those oflysine, ornithine, cysteine, serine, histidine, arginine, glutamic oraspartic acid (i.e. amines, alcohols, sulfhydryls, imidazoles,guanidines, carboxylates)) are suitably protected (using standardpeptide functional group protection) to eliminate their reactivity.Among unnatural side-chains, those that would react with the low-valentmetal complexes (e.g. aryl-halides, allylic esters, isothiocyanates, orisocyanates) also cannot be used.

The R″ group is crucial as this is the group that will typically be usedto “tag” or functionalize the polypeptide chains, and is the reason andadvantage for using this method. This group can be virtually anything,bearing in mind the chemical limitations of functional groups as listedabove for R′. Typically, this group will be a peptide, oligosaccharide,oligonucleotide, fluorescent molecule, polymer chain, small moleculetherapeutic, chemical linker to attach the polypeptide to a substrate,chemical linker to act as a sensing moiety, or reactive linker to couplethe polypeptide to larger molecules such as proteins, polysaccharides orpolynucleotides.

In preferred embodiments, N_(α)-allyloxycarbonyl-amino acid amides werereacted with zerovalent nickel complexes LNi(1,5-cyclooctadiene)(L=2,2′-bipyridine (bpy), 1,10-phenanthroline (phen),1,2-bis(dimethylphosphino)ethane (dmpe), and1,2-bis(diethylphosphino)ethane (depe)), to yield amido-amidatemetallacycles of the general formula: LNiNHC(R′)HC(O)NR″ (see Table 1below).

As shown in Table 2 (below), these complexes were found to initiatepolymerization of α-amino acid-N-carboxyanhydrides (NCAs) yieldingpolypeptides or block copolypeptides with defined molecular weights,narrow molecular weight distributions, and with quantitativeincorporation of the initiating ligand as an end-group. These initiatorsprovide a facile method to synthesize complex copolypeptides where thepolymer chain carboxy-terminus can be quantitatively functionalized witha wide range of substituents. These substituents can include, but arenot limited to: polymers (polystyrene, poly(ethylene oxide)), peptides,oligosaccharides, oligonucleotides, or other organic moieties.

The key feature utilized to connect these substituents to a polypeptidechain, and the only requirement of the substituents, is a primary aminegroup. This feature allows the preparation of complex polypeptidebiomaterials which have great potential applications in medicine (drugdelivery, tissue engineering). Specifically, the ability to incorporatechain-end functional groups (e.g. other polymers, fluorescent orradioactive tags, or biomolecules (peptide sequences, oligonucleotides,or oligo sachharides)) imparts these materials with highly desirablequalities for numerous biotechnological applications. For example, thismethodology can be used for labelling polypeptide chains to analyzechain mobility/location in vivo/in vitro. Moreover, incorporation ofendgroup functionalities, such as signaling or receptor groups, ontopolypeptide chains is essential for targeting of drug delivery complexesas well as substrate specific anchoring of these materials.

To form the desired amido-amidate nickelacycles, we conducted a reactionwhere N_(α)-Alloc-amino acid allyl amides were used as substrates (eq 7)

This reaction was not expected to be highly successful since theoxidative addition of allylic amides to nickel is without precedent(Collman, J. P.; Hegedus, L. S.; Norton, J. R.; Finke, R. G. Principlesand Applications of Organotransition Metal Chemistry 2nd Ed., UniversityScience, Mill Valley, 1987). A surprising result was therefore obtainedwhen the reaction of bpyNi(COD) with N_(α)-Alloc-L-leucine allyl amidewas found to produce an amido-amidate species in good yield (60%isolated). The product, however, was not the expected one, as evidencedby the lack of byproduct 1,5-hexadiene. The product nickelacycle wasfound to result from initial addition across the Alloc C—O bond,followed by a second addition across the N—H bond of the amide, not theallylic N—C bond (eq 7). As a result, the product metallacycle, 1,retained the allyl substituent on nitrogen, as determined by FAB/MS, ¹HNMR of the hydrolysis product from reaction with HCl, and ¹³C labelingstudies. The N—H addition was also verified by use of aN_(α)-2-hexenyloxycarbonyl-amino acid allyl amide in the reaction whichresulted in the formation of byproduct hexenes that were identified by¹³C {H} NMR.

The reaction of readily synthesized N_(α)-Alloc-amino acid allyl amideswith zerovalent nickel was found to be general for differentsubstituents (R′ and R″) and donor ligands, allowing the use of manycombinations of amino acids and primary amines in the construction ofinitiator complexes (Table 1). This method is therefore amenable toincorporation of a wide variety of end-group functionalities ontopolypeptides through amide linkages.

TABLE 1

Initiator

R′ R″ Yield (%) 1 bpy

69 (95) 2 phen

72 (94) 3 depe

42 (98) 4 depe

39 (97) 5 phen

59 (94) 6 phen

62 (96) Example initiators synthesized from different N-Alloc-α-aminoamides, bidentate ligands and Ni(COD)₂. a = isolated yield of initiator.Yield of crude product, as determined by FTIR spectroscopy, is given inparentheses.

At this point, it was necessary to determine if these functional groups,once attached to the initiating complex, were then quantitativelyincorporated as end-groups on polypeptide chains. Polymerizations ofγ-benzyl-L-glutamate NCA (Glu NCA) using nickel complexes containingdifferent bidentate donor ligands revealed that alkyl phosphine ligands(dmpe and depe) promoted the most efficient initiation. These initiatorswere able to prepare block copolypeptides of defined sequence andcomposition (Table 2).

TABLE 2 Synthesis of polypeptides and block copolypeptides using 4 (seeTable 1) in DMF at 20° C. Lys NCA = ε-CBZ-L-lysine-N-carboxyanhydride.First Diblock segment^(b) Copolymer^(c) First Second M_(w)/ M_(w)/ YieldMonomer^(a) Monomer^(a) M_(n) M_(n) M_(n) M_(n) (%)^(d) 25 Glu-NCA none5190 1.37 — — 88 50 Glu-NCA nono 13450# 1.31 — — 83 200 Glu-NCA none49340  1.24 — — 90 25 Glu-NCA 71 Lys-NCA 5190 1.37 28880 1.18 75 25Lys-NCA 87 Glu-NCA 8760 1.06 25600 1.13 77 ^(a)= First and secondmonomers added stepwise to the initiator; number indicates equivalentsof monomer per 4. ^(b)= Molecular weight and polydispersity index afterpolymerization of the first monomer. ^(c)= Molecular weight andpolydispersity index after polymerization of the second monomer. ^(d)=Total isolated yield of polypeptide or block copolypeptide.

It was also found that the initiators could be used for polymerizationwithout isolation from the crude reaction mixture. This feature greatlysimplifies the use of these complexes, which are near-quantitativeyield, but can be tedious to isolate from the reaction solvent. Hence,polymerizations were conducted using either isolated or in situinitiators with no noticeable differences in results.

Concerning the degree of functionalization of the polymers, reaction ofinitiator 4 (see Table 1) with one equivalent ofcis-5-norbornene-endo-2,3-dicarboxylic anhydride, which should add tothe initiator like an NCA monomer but not form polymer, followed byhydrolysis of the product with HCl, resulted in complete consumption of4 to yield the addition product (eq 8). The anhydride IR stretch of thestarting material (1780 cm⁻¹) was observed to completely disappear overthe course of the reaction. The only amino acid containing compoundpresent after hydrolysis was the coupled product (FAB MS: MH⁺: 323.8calc'd, 323 found). No unreacted monopeptide from hydrolysis ofunreacted 4 was detected, showing that all metal centers were active.

Furthermore, polymerization studies using initiator 3 (see Table 1) gavepolypeptides with a 1-naphthyl end-group. These end-groups were thenquantitated using fluorescence spectroscopy, which showed that thenumber of end-groups increased commensurately with the number of polymerchains. These fluorescent tags are useful for monitoring polypeptidelocation and mobility, desirable for applications such as the monitoringof drug delivery complexes in vitro (Singhal, A.; Huang, L. in GeneTherapeutics: Methods and Applications of Direct Gene Transfer, J. A.Wolff Ed., Birkhauser, Boston, 1994).

Finally, MALDI-MS analysis of phenylglycine oligomers prepared using anickel complex containing a leucine isoamylamide initiating grouprevealed that nearly all chains were end-functionalized with the leucineresidue of the initiator. (See FIG. 7) Only very small peaks wereobserved for non-functionalized oligo(phenylglycines), indicating thatthe degree of chain functionalization was greater than 98%.

Amido-Containing Metallacycles

Another embodiment of the invention entails a five or six memberedamido-containing metallacycle comprising molecules of the generalformula:

wherein “M” is a low valent transition metal capable of undergoing anoxidative addition reaction, “L” is an electron donor such as a Lewisbase and “R#” comprises any organic substituent not bearing free amine,hydroxyl, carboxylic acid, sulfhydryl, isocyanate, imidazole, or otherhighly protic or nucleophilic functionality. These functionalities maybe present, however, if suitably chemically protected to render themunreactive as protic sources or nucleophiles. Effective R substituentson the above structures exhibit a number of properties. For example, asdisclosed in the examples below, the R substituents on the abovestructures are typically encompassed by the structures of the side chainsubstituents of amino acids or derivatives thereof. In particular, inmost cases, R1 (and R4 in the center structure) is a proton.Independently, each of R2 and R3 (and R5 and R6) are typically selectedfrom the side chain substituents of amino acids. Typically, one of thesubstituents (such as R1) is a proton (H), while the others can bedifferent side chain group of a specific amino acid. The placement ofthe proton (as either R2 or R3) is determined by the amino acid being ofthe L or D configuration. The side chain will be one of those from thefamily of naturally occurring L- or D-amino acids, or synthetic aminoacids or derivatives thereof. Naturally occurring L- or D-amino acids(e.g. alanine, arginine, asparagine, aspartic acid, g-carboxyglutamate,cysteine, glutamic acid, glutamine, glycine, histidine, hydroxyproline,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine and valine) and synthetic amino acids orderivatives thereof are well known in the art. The side-chains of aminoacids bearing polar functional groups (e.g. NH₂, COOH, SH, imidazole)can be blocked with standard peptide protecting groups.

In a preferred embodiment, the metal is a group VIII transition metaland the donor ligand(s) can be any of those given in Table 7. In anotherpreferred embodiment, the metal is nickel and the donor ligand is a2,2′-bipyridyl (bipy) moiety. In another preferred embodiment, the R2 orR3 group comprises an amino acid side chain selected from the groupconsisting of side-chain protected NCA formed from arginine, asparagine,aspartic acid, cysteine, glutamic acid, glutamine, histidine, lysine,methionine, serine, threonine, tryptophan, and tyrosine or an amino acidside chain selected from the group consisting of side-chain NCA formedfrom alanine, glycine, isoleucine, leucine, phenylalanine, proline andvaline.

Ruthenium Initiators for Synthesis of Polypeptides and BlockCopolypeptides

We recently developed a new class of α-amino acid-N-carboxyanhydride(NCA) polymerization initiators based on p-cymene ruthenium(II) amidometallacycle complexes. These initiators differ from our earlierdisclosed initiators (describe above) in that they do not contain theamido-amidate metallacycle structure, which was found to be the keyfunctional unit required for controlled NCA polymerization. Althoughdiffering in structure, these new ruthenium initiators were recognizedto possess all the required features for controlled NCA polymerizationdetermined previously, namely: a nucleophilic alkyl amido group,stabilized by a rigid chelate, and a proton-accepting group on the otherend of the metallacycle (a sulfonamide) that allows the chain-end tomigrate off the metal, but which is also non-nucleophilic.

It is worth noting that the ruthenium complex is monomeric in thesolid-state and does not dimerize through the lone pairs on the amidogroups. This is a substantial improvement over our cobalt and nickelamido-containing metallacycles, which aggregate strongly. The rutheniumcomplex was found to be an efficient initiator for the controlledpolymerization of NCAs, including preparation of block copolypeptides.Furthermore, initiation efficiency was found to be less solventdependent than our earlier systems, commensurate with the ability of theruthenium initiator to avoid aggregation. These results show thatamido-containing metallacycles can be prepared with chemical structuresother than the amido-amidate metallacycle, where the amidate group canbe replaced with other chemical functionalities (e.g. sulfonamide), andresult in stable and effective initiators for controlled NCApolymerizations. Improvements in catalysis based on these new systemscan be expected to allow the preparation of better complex polypeptidebiomaterials (narrower molecular weight distributions, better definedand shorter domains of amino acids sequences) which have great potentialapplications in medicine (drug delivery, tissue engineering,therapeutics).

We conducted NCA polymerization studies with p-cymene ruthenium(II)amido complex (1), which is a known compound that is prepared bytreating the corresponding amino chloride complex with aqueous base (eq9). While this is not an amido-amidate metallacycle, 1 was recognized topossess all the required features for controlled NCA polymerization.This complex contains a nucleophilic alkyl amido group, stabilized by arigid chelate, and a proton-accepting group on the other end of themetallacycle (the sulfonamide, N-ts group) that allows the chain-end tomigrate off the metal, but which is also non-nucleophilic. It is worthnoting that the 16e complex 1 is monomeric in the solid-state and doesnot dimerize through the lone pairs on the amido groups.¹ This is asubstantial improvement over our cobalt and nickel amido-containingmetallacycles, which aggregate strongly. When mixed with 3 equivalentsof the donor ligand PMe₃, complex 1 was found to be an efficientinitiator for the controlled polymerization of NCAs, includingpreparation of block copolypeptides (Table 1).

TABLE 1 I = 1 + 3 PMe₃ for all polymerizations. M = Glu NCA monomer. Thediblock copolymer was prepared in DMF by adding 50 total equivalents ofmonomer in two equal batches. Addition of the second batch was performedafter all of the first batch was consumed. THF DMF [M]/[I] M_(n)M_(w)/M_(n) Yield (%) M_(n) M_(w)/M_(n) Yield (%) 25 12 000 1.26 95 5500 1.14 92 50 24 100 1.17 96 12 000  1.18 93 Diblock 1st addition 2ndaddition 25 + 25  4 500 1.13 NA 9 000 1.15 96

Furthermore, initiation efficiency was found to be less solventdependent than our earlier systems, commensurate with the ability of 1to avoid aggregation. These results show that amido-containingmetallacycles can be prepared with chemical structures other than theamido-amidate metallacycle, where the amidate group can be replaced witha variety of chemical functionalities, and result in stable andeffective initiators for controlled NCA polymerizations. Improvements incatalysis based on these new systems can be expected to allow thepreparation of better complex polypeptide biomaterials (narrowermolecular weight distributions, better defined and shorter domains ofamino acids sequences) which have great potential applications inmedicine (drug delivery, tissue engineering, therapeutics).

The success of the ruthenium initiator described above shows that manyother initiator structures besides the amido-amidate (or amido-alkyl)metallacycles described above can be used for controlled NCApolymerizations. The key features that appear to be required forsuccessful initiator formation are those that were previouslyidentified, namely: a metallacycle containing a nucleophilic alkyl amidogroup, stabilized by a rigid chelate, and a proton-accepting group onthe other end of the metallacycle that allows the chain-end to migrateoff the metal, but which is also non-nucleophilic. The new feature shownin this disclosure is that the “proton-accepting group” can be somethingdifferent that the originally discovered amido-amidate unit. Thisoriginal structure, and some possible alternatives that have potentialto form good initiators, are shown in FIG. 1. Note that manyamido-containing metallacycles can be functionally equivalent goodinitiators, such as the following new structures:

wherein R2, R3, R5, and R6 are each independently hydrogen or anyorganic substituent not bearing free amine, hydroxyl, carboxylic acid,sulfhydryl, isocyanate, imidazole, or other highly protic ornucleophilic functionality, e.g. C1-C12 alkyl or aryl groups such asphenyl. Similarly, R4 and R7 are each any organic substituent notbearing free amine, hydroxyl, carboxylic acid, sulfhydryl, isocyanate,imidazole, or other highly protic or nucleophilic functionality. A mostpreferred Lewis base donor ligand, L, is p-cymene and the most preferredlow valent transition metal, M, is ruthenium.

Adding NCA monomers

A related embodiment of the invention consists of a method of adding anaminoacid-N-carboxyanhydride (NCA) to a polyaminoacid chain having anamido containing metallacycle end group comprising combining the NCAwith the polyaminoacid chain so that the NCA is added to thepolyaminoacid chain. In a preferred embodiment of this method, the amidocontaining metallacycle end group is of a formula as follows:

wherein M is the low valent transition metal;

L is the Lewis Base ligand;

R1 comprises a constituent found in a side chain of an amino acid (e.g.a hydrogen for glycine or a methyl group for alanine etc.) selected fromthe group consisting of alanine, arginine, asparagine, aspartic acid,cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine and valine;

R2 comprises a constituent found in a side chain of an amino acidselected from the group consisting of alanine, arginine, asparagine,aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine and valine;

R3 comprises a constituent found in a side chain of an amino acidselected from the group consisting of alanine, arginine, asparagine,aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine and valine; and

R4 is the polyaminoacid chain.

In a preferred embodiment of the method of adding anaminoacid-N-carboxyanhydride (NCA) to a polyaminoacid chain having anamido containing metallacycle end group, the metal group of the amidocontaining metallacycle is a transition metal selected from the groupconsisting of nickel, palladium, platinum, cobalt, rhodium, iridium andiron and the Lewis Base ligand is selected from the group consisting ofpyridyl ligands, diimine ligands, bisoxazoline ligands, alkyl phosphineligands, aryl phosphine ligands, tertiary amine ligands, isocyanideligands, and cyanide ligands. In specific embodiments of the invention,the NCA is an a-aminoacid-N-carboxyanhydride selected from the groupconsisting of alanine, arginine, asparagine, aspartic acid, cysteine,glutamic acid, glutamine, glycine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine and valine.

Polymerization Reactions

Another embodiment of the invention disclosed herein entails a method ofpolymerizing aminoacid-N-carboxyanhydride monomers by combining a NCAmonomer with an initiator molecule complex comprised of a low valenttransition metal-Lewis Base ligand. A specific embodiment of theinvention disclosed herein entails a method of polymerizingaminoacid-N-carboxyanhydride monomers having a ring with a O—C₅ and aO—C₂ anhydride bond. The method consists of combining a first NCAmonomer with an initiator molecule complex. The complex is comprised ofa low valent metal capable of undergoing an oxidative addition reaction,wherein the oxidative addition reaction formally increases the oxidationstate by two electrons, and an electron donor comprising a Lewis base.The initiator molecule opens the ring of the first NCA through oxidativeaddition across either the O—C₅ or O—C₂ anhydride bond and combines witha second NCA monomer to form an amido-containing metallacycle. A thirdNCA monomer then combines with the amido containing metallacyle so thatthe amido nitrogen of the amido containing metallacyle attacks thecarbonyl carbon of the NCA. The NCA is then added to the polyaminoacidchain, and the amido containing metallacyle is regenerated for furtherpolymerization.

In a preferred embodiment of the invention, the efficiency of theinitiator is controlled by allowing the reaction to proceed in a solventselected for its ability to influence the reaction. In a specificembodiment of the invention, the solvent is selected from the groupconsisting of ethyl acetate, toluene, dioxane, acetonitrile, THF andDMF.

As illustrated in Example 5 below, the efficiency of various initiatorscan be analyzed through polymerization experiments with Glu-NCA. Theresulting polymer, PBLG, is α-helical in many solvents, has beenextensively studied, and is readily characterized. H. Block,Poly(g-benzyl-L-glutamate) and Other Glutamic Acid Containing Polymers,Gordon and Breach, New York, (1983). Number-average molecular weight ofPBLG samples formed using bipyNi(COD) in DMF was found to increaselinearly as a function of the initial monomer to initiator ratios,indicating the absence of chain-breaking reactions. L. J. Fetters,“Monodisperse Polymers” in Encyclopedia of Polymer Science andEngineering 2nd Ed., Wiley-Interscience, New York, 10:19-25 (1987); O.Webster, “Living Polymerization Methods” Science, 251:887-893 (1991).Such control over polypeptide molecular weight is a substantialimprovement over conventional NCA polymerization systems (see FIG. 1).The polymers possessed narrow molecular weight distributions(M_(w)/M_(n)=1.05-1.15) and were obtained in excellent yields (95-99%isolated). Kinetic analysis also showed that the polymerizations werewell behaved. The polymerizations were first order in monomerconcentration over 4 half-lives in DMF (k_(obs)=2.7(1)×10⁻⁴ s⁻¹ at 298K;[bipyNi(COD)]=0.67 mM) showing none of the complexities of traditionalNCA polymerizations. Our initiating system displays all of thecharacteristics of a living chain growth process for Glu-NCA. Analysisof other NCA monomers (e.g. e-carbobenzyloxy-L-lysineN-carboxyanhydride, Lys-NCA) also yielded controlled polymerizations,illustrating the general utility of our initiating system forpreparation of well-defined block copolypeptides with a variety ofarchitectures.

Making Block Copolypeptides

Block copolymers have played an important part in materials science andtechnology because they allow the effective combination of disparateproperties in a single material. Block copolymers of styrene and dienes,for example, are rubbers at room temperature (a characteristic of thepolydiene phase) but can be moulded at temperatures above the glasstransition of the polystyrene phase. This distinguishes the blockcopolymers from most conventional rubbers, which must be chemicallycross-linked (vulcanized) in order to withstand the stresses that theyencounter in use. Because chemical cross-linking is irreversible, itmust be done while making the final part; and cross-linked rubber isdifficult to reprocess. The ‘cross-linking’ step for styrene-diene blockcopolymers is instead a physical association of chains in the glassypolystyrene domains: the tendency of the two different chain sections toclump together, like with like. This association is robust enough tobear loads at room temperature, but is readily reversible upon heating.

Well-defined block copolymers assemble spontaneously into a variety ofintriguing nanostructures, and other, aligned nano-structure arrays canbe made using fluid flow or other fields. Z. R. Chen, et al., Science,277:1248-1253 (1997). Because of this, block copolymers have enjoyedgreat commercial success, as well as the ardent attentions of polymerphysicists. But block copolymers of amino acids have been littlestudied, largely because our synthetic methods do not have fine enoughcontrol to produce well-defined structures. F. Cardinauz, et al.,Biopolymers, 16:2005-2028 (1977). The same is true of the synthesis ofblock copolypeptides for use as biomaterials or as selectivemembranes—the potential advantages of the protein-like architectureshave remained unrealized for want of adequate synthetic tools. Thedisclosed methods and compositions promise to change that. By treatingthe monomer of interest with an initiator such as the zero-valent nickelcomplex bipyNi (COD), where bipy is 2,2′-bipyridyl and COD is1,5-cyclooctadiene and adding NCAs yields an intermediate molecule thatcan be isolated and that remains active towards further ring-openingpolymerization, and the target polypeptide can be prepared withessentially 100 percent yield.

One embodiment of the invention provides a method of making a blockcopolypeptide consisting of combining an amount of a firstaminoacid-N-carboxyanhydride monomer with an initiator moleculecomprising a low valent transition metal-Lewis Base ligand complex sothat a polyaminoacid chain is generated and then combining an amount ofa second aminoacid-N-carboxyanhydride monomer with the polyaminoacidchain so that the second aminoacid-N-carboxyanhydride monomer is addedto the polyaminoacid chain. In a preferred embodiment of this method,the initiator molecule combines with the firstaminoacid-N-carboxyanhydride monomer to form an amido containingmetallacycle intermediate of the general formula:

wherein M is the low valent transition metal;

L is the Lewis Base ligand;

and each of R1 and R2 and R3 independently consist of a side chain of anamino acid selected from the group consisting of alanine, arginine,asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, threonine, tryptophan, tyrosine and valine; and

R4 is the polyaminoacid chain.

In a highly preferred embodiment of this method of making a blockcopolypeptide, the low valent transition metal is selected from thegroup consisting of nickel, palladium, platinum, cobalt, rhodium,iridium and iron. In another preferred embodiment of this method, theLewis Base ligand is selected from the group consisting of pyridylligands, diimine ligands, bisoxazoline ligands, alkyl phosphine ligands,aryl phosphine ligands, tertiary amine ligands, isocyanide ligands andcyanide ligands.

In yet another preferred embodiment of this method, the firsta-aminoacid-N-carboxyanhydride monomer is an NCA is ana-aminoacid-N-carboxyanhydride selected from the group consisting ofside-chain protected NCA formed from arginine, asparagine, asparticacid, cysteine, glutamic acid, glutamine, histidine, lysine, methionine,serine, threonine, tryptophan, and tyrosine or an amino acid side chainselected from the group consisting of side-chain NCA formed fromalanine, glycine, isoleucine, leucine, phenylalanine, proline andvaline.

Block Copolypeptides

Another embodiment of the invention entails a block copolypeptidewherein the number of overall monomer units (residues) in the blockcopolypeptide is greater than about 100; and the distribution ofchain-lengths in the block copolymer composition is at least about1.01<Mw/Mn<1.25, where Mw/Mn=weight average molecular weight divided bynumber average molecular weight. In one embodiment, the blockcopolypeptide has 10 consecutive identical amino acids per block. In apreferred embodiment, the block copolypeptide is composed of amino acidcomponents g-benzyl-L-glutamate and e-carbobenzyloxy-L-lysine. Inanother preferred embodiment, the copolypeptide is apoly(e-benzyloxycarbonyl-L-Lysine-block-g-benzyl-L-glutamate),PZLL-b-PBLG, diblock copolymer. In yet another preferred embodiment thecopolypeptide is apoly(g-benzyl-L-glutamate-block-e-benzyloxycarbonyl-L-Lysine-block-g-benzyl-L-glutamate)triblock copolymer. In related embodiments, the number of consecutivemonomer units (residues) in the block copolypeptide is greater thanabout 50 or 100 or 500 or 1000 (see e.g. the examples disclosed inTables 1 and 4). In another related embodiment, the total number ofoverall monomer units (residues) in the block copolypeptide is greaterthan about 200, or greater than about 500, or greater than about 1000(see e.g. the examples disclosed in Tables 1 and 4).

Illustrative embodiments of the invention that are disclosed in theexamples below include diblock copolymers composed of amino acidcomponents g-benzyl-L-glutamate and e-carbobenzyloxy-L-lysine. Thepolymers were prepared by addition of Lys-NCA to bipyNi(COD) in DMF toafford living poly(e-carbobenzyloxy-L-lysine), PZLL, chains withorganometallic end-groups capable of further chain growth. Glu-NCA wasadded to these polymers to yield the PBLG-PZLL block copolypeptides. Theevolution of molecular weight through each stage of monomer addition wasanalyzed using gel permeation chromatography (GPC) and data are given inTable 1 in Example 3 below. Molecular weight was found to increase asexpected upon growth of each block of copolymer while polydispersityremained low, indicative of successful copolymer formation. A. Noshay,et al., Block Copolymers, Academic Press, New York, (1977).

The chromatograms of the block copolypeptides showed single sharp peaksillustrating the narrow distribution of chain lengths (See FIG. 2).Copolypeptide compositions were easily adjusted by variation of monomerfeed compositions, both being equivalent. Successful preparation ofcopolypeptides of reverse sequence (i.e. PZLL-PBLG) and of triblockstructure (e.g. PBLG_(0.39)-b-PZLL_(0.22)-b-PBLG_(0.39); M_(n)=256,000,M_(w)/M_(n)=1.15) illustrate the sequence control using the nickelinitiator.

Block copolymerizations were not restricted to the highly solublepolypeptides PBLG and PZLL. Copolypeptides containing L-leucine andL-proline, both of which form homopolymers which are insoluble in mostorganic solvents (e.g. DMF) have also been prepared. Data for thesecopolymerizations are given in Table 1 in Example 2 below. Because ofthe solubilizing effect of the PBLG and PZLL blocks, all of the productswere soluble in the reaction media indicating the absence of anyhomopolymer contaminants. The block copolymers containing L-leucine werefound to be strongly associating in 0.1M LiBr in DMF, a good solvent forPBLG and PZLL. Once deprotected, the assembly properties of thesematerials are expected make them useful as tissue engineering scaffolds,drug carriers, and morphology-directing components in biomimeticcomposite formation.

As discussed above, embodiments of the present invention provide anumber of novel methods and compositions for the generation ofpolypeptides having varied material properties. The description andillustrative examples disclosed herein provide a number of exemplaryembodiments of the invention. Specific embodiments of the inventioninclude methods of adding aminoacid-N-carboxyanhydrides (NCAs) topolyaminoacid chains by exposing the NCA to solutions containingpolyaminoacid chains having an amido amidate metallacyle end groups andreacting the NCA with the amido amidate metallacyle end group so thatthe NCA is added to the polyaminoacid chain. Addition embodimentsinclude methods of controlling the polymerization ofaminoacid-N-carboxyanhydrides by reacting NCAs with initiator moleculesand allowing initiator complexes to regioselectively open the ring ofthe NCAs through oxidative addition across the O—C₅ or O—C₂ anhydridebond resulting a controlled polypeptide polymerization. Otherembodiments include methods for making amido-containing metallacyclesare disclosed herein. Additional embodiments of the invention includecompositions for use in peptide synthesis and design including five andsix membered amido-containing metallacycles and block copolypeptides.

In addition to block copolypeptides, a variety of other related ofpolypeptides can be generated utilizing the methods disclosed hereinwherein an initiator molecule combines with a firstaminoacid-N-carboxyanhydride monomer to form an amido containingmetallacycle intermediate of the five and six ring formulae disclosedherein. For example polypeptides can be generated where the domains caneither be repeats (2 or greater) of identical amino acids, or can berepeats (2 or greater) of mixtures of distinct amino acids, or acombination of the two. The number, length, order, and composition ofthese domains can vary to include all possible amino acids in any numberof repeats. Preferably the total number of overall monomer units(residues) in these polypeptides having segregated domains of mixedmonomers is greater than 100 and the distribution of chain-lengths inthe polypeptide is about 1.01<Mw/Mn<1.25, where Mw/Mn=weight averagemolecular weight divided by number average molecular weight.

An illustrative example of such a polypeptide could contain a sequenceof, for example, 50 residues of leucine in one domain, followed by astatistical mixture of 20 valines and 20 glycines as the second domain,followed finally by a third domain of 40 phenylalanines. Suchpolypeptides are substantially different than “statistically random”copolymers where the entire polypeptide is composed of statisticalmixtures of amino acids in the chains, and there are no strict blockdomains. One difference is that these polypeptides have segregateddomains where one statistical mixture will be separated from the others.For example in a statistical copolymer, the amino acids will bedistributed statistically (basically at random) along the entirepolypeptide chain. In contrast, using the methods disclosed herein apolypeptide chain can be constructed such that in one domain there willbe a statistical mixture of leucine and glycine, followed by a seconddomain consisting of a statistical mixture of glycine and valine.Although both copolymers have statistical mixtures of residues along thechain, these polypeptide differ in that the valine and leucine residuesare segregated into separate domains.

The living polymerization methods for NCAs that are disclosed hereinwill lead to various polypeptides and block copolypeptides having avariety of new and useful properties. In this context, the disclosureprovided herein demonstrates the successful synthesis of such materials,and creates a new family of polypeptides that link combination ofacidic, basic and hydrophobic domains, all with excellent control ofmolecular architecture. The prospects for application in biomedicalengineering, drug delivery and selective separations are excellent. Inparticular these features allow the preparation of complex polypeptidebiomaterials which have potential applications in biology, chemistry,physics, and materials engineering. Potential applications includemedicine (drug delivery, tissue engineering), “smart”₂ hydrogels(environmentally responsive organic materials), and in organic/inorganicbiomimetic composites (artificial bone, high performance coatings).

Self-Assembling Amphiphilic Block Copolypeptides for BiomedicalApplications

Yet another embodiment of the present invention entails the synthesis ofamphiphilic block copolypeptides, which contain at least one watersoluble block polypeptide (“soluble block”) conjugated to awater-insoluble polypeptide domain (“insoluble block”). The overall mole% composition of the insoluble block(s) can range from 3-60% of thetotal copolymer. Preferably, the soluble block has about 30 to 100 molepercent identical amino acid residues having either charged oroligo(ethyleneglycol)-conjugated side chains.

The amphiphilic block copolypeptides of the present invention containone or more “soluble blocks.” The soluble block(s) of the copolymers cancontain some finite fraction of amino acid components with chargedside-chains, with the amino acids belonging to the group: glutamic acid,aspartic acid, arginine, histidine, lysine, or ornithine. They can alsocontain up to a maximum 99 mole % of the amino acidsoligo(ethyleneglycol). The soluble block includes oligo (ethyleneglycol)terminated amino acid (EG-aa) residues. Preferred oligo(ethyleneglycol)functionalized amino acid residues include EG-Lys, EG-Ser, EG-Cys, andEG-Tyr. A most preferred soluble block consists of oligo(ethyleneglycol)terminated poly(lysine).

The amphiphilic block copolypeptides of the present invention alsocontains at least one “insoluble block,” which is covalently linked tothe soluble block. The insoluble block can contain a variety of aminoacids residues or mixtures thereof, including the naturally occurringamino acids, ornithine, or blocks consisting entirely of one or moreD-isomers of the amino acids. However, the insoluble block willtypically be composed primarily of nonionic amino acid residues, whichgenerally form insoluble high molecular weight homopolypeptides. Inpreferred embodiments about 60 to about 100 mole percent of theinsoluble block is comprised of nonionic amino acids. Such nonionicamino acids include, but are not limited to phenylalanine, leucine,valine, isoleucine, alanine, serine, threonine and glutamine. In anotherpreferred embodiment, any given insoluble block will usually contain 2-3different kinds of amino acid components in a statistically randomsequences with mixtures of leucine/phenylalanine and leucine/valinebeing preferred. In these preferred copolymers, the composition ofleucine in the insoluble domain ranges from 25-75 mole % when mixed withphenylalanine and ranges from 60-90% when mixed with valine.

Vesicle Formation

The amphiphilic block copolypeptides of the present invention areincluded in methods and compositions of matter, which form vesicularstructures in aqueous solutions. The method consists of suspending theamphiphilic block copolypeptides in an aqueous solution so that thecopolypeptides spontaneously self assemble into vesicles. Accordingly,the vesicle-containing compositions are comprised of the amphiphilicblock copolypeptides of the present invention and water.

The vesicles range in controllable size from microns to less than ahundred nanometers in diameter, similar to liposomes yet more robust,which make them potentially valuable for biomedical/biotechnologicalapplications (i.e. drug delivery). In one specific embodiment, smallervesicles having a diameter of about 50 nm to about 500 nm can be formedby sonicating the suspension of larger vesicles. In addition, thediameter of the vesicles can be controlled by adjusting the length andamino acid composition of the amphiphilic block copolypeptide (see,e.g., the examples below).

EXAMPLES

The following examples are offered by way of illustration and not by wayof limitation. The disclosures of all citations in the specification areexpressly incorporated herein by reference into this application inorder to more fully describe the state of the art to which thisinvention pertains.

Example 1 Methods Using Amino Acid Derived Metallacycles: Intermediatesin Metal Mediated Polypeptide Synthesis

General Experimental Protocols and Reagents

Infrared spectra were recorded on a Perkin Elmer 1605 FTIRSpectrophotometer calibrated using polystyrene film. Tandem gelpermeation chromatography/light scattering (GPC/LS) was performed on aSpectra Physics Isochrom liquid chromatograph pump equipped with a WyattDAWN DSP light scattering detector and Wyatt Optilab DSP. Separationswere effected by 10⁵ Å and 10³ Å Phenomenex 5μ columns using 0.1M LiBrin DMF eluent at 60° C. Optical rotations were measured on a PerkinElmer Model 141 Polarimeter using a 1 mL volume cell (1 dm length). NMRspectra and bulk magnetic susceptibility measurements (Evans method)were measured on a Bruker AMX 500 MHz spectrometer. [D. F. Evans, J.Chem. Soc., 2003-2009 (1959); J. K. Becconsal, J. Mol. Phys., 15:129-135(1968)]. C, H, N elemental analyses were performed by theMicroanalytical Laboratory of the University of California, BerkeleyChemistry Department. Chemicals were obtained from commercial suppliersand used without purification unless otherwise stated. (COD)₂Ni wasobtained from Strem Chemical Co., and ¹³C₁-L-leucine and ¹³C-phosgenewere obtained from Cambridge Isotope Labs. L-leucine isoamylamidehydrochloride, g-benzyl-L-glutamate NCA and L-leucine NCA were preparedaccording to literature procedures. M. Bodanszky, et al., The practiceof Peptide Synthesis, 2nd Ed., Springer, Berlin/Heidelberg, (1994); E.R. Blout, et al., J. Am. Chem. Soc., 78:941-950 (1956); H. Kanazawa, etal., Bull. Chem. Soc. Jpn., 51:2205-2208 (1978). Hexanes, THF, andTHF-d₈ were purified by distillation from sodium benzophenone ketyl. DMFand DMF-d₇ were purified by drying over 4 Å molecular sieves followed byvacuum distillation.

(S)-[NiNHC(H)RC(O)NCH₂R]_(x), R=—CH₂CH₂C(O)OCH₂C₆H₅; 1

In the dry box, Glu NCA (15 mg, 0.058 mmol) was dissolved in THF (0.5mL) and added to a stirred homogeneous mixture of PPh₃ (31 mg, 0.12mmol) and (COD)₂Ni (16 mg, 0.058 mmol) in THF (1.5 mL). The red/brownsolution was stirred for 24 hours, after which the solvent was removedin vacuo to leave a dark red oily solid. This was extracted with hexanes(3×5 mL) to yield a red/brown hexanes solution and a yellow solid.Evaporation of the hexanes solution gave a red oil containing(PPh₃)₂Ni(CO)₂ [IR (THF): 2000, 1939 cm⁻¹ (nCO, vs); 18 mg; Literature:IR (CH₂ClCH₂Cl): 1994, 1933 cm⁻¹)], and drying of the solid gave theproduct as a yellow powder (10 mg, 75% yield). J. Chatt, et al., J.Chem. Soc., 1378-1389 (1960). An ¹H NMR spectrum could not be obtainedin THF-d₈, most likely because of paramagnetism of the complex (onlybroad lines for the benzyl ester groups were observed). m_(eff) (THF,293 K)=1.08 m_(B). Osmotic molecular weight in THF (vs. ferrocene; ca. 7mg/mL): 910 g/mol; this corresponds to a degree of aggregation of 1.94.IR (THF): 3281 cm⁻¹ (nNH, s br), 1734 cm⁻¹ (nCO, ester, vs), 1577 cm⁻¹(nCO, amidate, vs). Anal. calcd. for NiC₂₃H₂₆N₂O₅: 58.87% C, 5.59% H,5.96% N. found: 59.07% C, 5.67% H, 5.56% N. [a]_(D) ²⁰ (THF,c=0.0034)=−71.

(S)-Cl⁻⁺H₃NC(H)RC(O)NHCH₂R, R=—CH₂CH(CH₃)₂

In the dry box, L-leucine NCA (9.2 mg, 0.058 mmol) was dissolved in THF(0.5 mL) and added to a stirred homogeneous mixture of PPh₃ (31 mg, 0.12mmol) and (COD)₂Ni (16 mg, 0.058 mmol) in THF (1.5 mL). The red/brownsolution was stirred for 24 hours, after which the solvent was removedin vacuo to leave a dark red oily solid. This was extracted with coldhexanes (0° C., 3×2 mL) to yield a red/brown hexanes solution and a paleorange solid. Evaporation of the hexanes solution gave a red oilcontaining (PPh₃)₂Ni(CO)₂ [IR (THF): 2000, 1939 cm⁻¹ (nCO, vs); 17 mg],and drying of the solid gave an orange powder which could be purified byprecipitation from THF/hexanes to give (S)-[NiNHC(H)RC(O)NCH₂R]_(x),R=—CH₂CH(CH₃)₂ as a yellow powder (6 mg, 80% yield). An ¹H NMR spectrumcould not be obtained in THF-d₈, most likely because of paramagnetism ofthe complex. IR (THF): 3290 cm⁻¹ (nNH, s br), 1580 cm⁻¹ (nCO, amidate,vs). [a]_(D) ²⁰ (THF, c 0.001)=−185.

This product was dissolved in THF (5 mL) in a round bottom Schlenk flaskin the dry box. The flask was placed under N₂ atmosphere on a Schlenkline and HCl (90 mL of a 1.0M solution in Et₂O) was then added. Theyellow solution turned orange and then became hazy as it slowly turnedgreen. After 2 h, the solvent was removed in vacuo to leave a greengummy solid. This solid was extracted with D₂O to isolate the amino acidcontaining products. The single isolated compound (4 mg, 73%) was foundto be identical to an authentic sample of L-leucine isoamylamidehydrochloride (FIG. 1). ¹H NMR (D₂O): d 3.94 (t,NH₃CH(CH₂CH(CH₃)₂)C(O)—, 1H, J=7.5 Hz), 3.33, 3.14 (dm, —C(O)NHCH₂CH₂CH(CH₃)₂, 2H, J_(gem)=107 Hz, J_(mult)=6 Hz, 13 Hz), 1.72 (dd,NH₃CH(CH ₂CH(CH₃)₂)C(O)—, 2H, J=6 Hz, 7 Hz), 1.68 (m,NH₃CH(CH₂CH(CH₃)₂)C(O)—, 1H, J=7 Hz), 1.63 (m, —C(O)NHCH₂CH₂CH(CH₃)₂,1H, J=7 Hz), 1.43 (ddd, —C(O)NHCH₂CH ₂CH(CH₃)₂, 2H, J=7 Hz), 0.98, 0.96(dd, NH₃CH(CH₂CH(CH ₃)₂)C(O)—, 6H, J=6 Hz), 0.92, 0.90 (dd,—C(O)NHCH₂CH₂CH(CH ₃)₂, 6H, J=6 Hz). [a]_(D) ²⁰ (THF, c=0.0033)=+10.3.Authentic Sample: [a]_(D) ²⁰ (THF, c=0.0033)=+10.5.

Reaction of (PPh₃)₂Ni(COD) with ¹³C₂-L-Leucine NCA

The procedure given above for the reaction using unlabeled L-leucine NCAwas followed exactly, except for the substitution of ¹³C₂-L-Leucine NCA[prepared from L-leucine and O¹³CCl₂; IR (CHCl₃): 3299 cm⁻¹ (nNH, s br),1836, 1745 cm⁻¹ (nCO, anhydride, vs); ¹³C {¹H} NMR (THF-d₈): d 152 (s,—NC(O)O—]. The product was extracted with cold hexanes (0° C., 3×2 mL)to yield a red/brown hexanes solution and a pale orange solid.Evaporation of the hexanes solution gave a red oil containing(PPh₃)₂Ni(CO)₂ [IR (THF): 2000, 1939 cm⁻¹ (nCO, vs)], and drying of thesolid gave an orange powder which could be purified by precipitationfrom THF/hexanes to give (S)-[NiNHC(H)RC(O)NCH₂R]_(x), R=—CH₂CH(CH₃)₂ (5mg, 66% yield). IR (THF): 3288 cm⁻¹ (nNH, s br), 1580 cm⁻¹ (nCO,amidate, vs).

Reaction of (PPh₃)₂Ni(COD) with ¹³C₅-L-Leucine NCA

The procedure given above for the reaction using unlabeled L-leucine NCAwas followed exactly, except for the substitution of ¹³C₅-L-Leucine NCA[prepared from ¹³C₁-L-leucine and OCCl₂; IR (KBr): 3308 cm⁻¹ (nNH, sbr), 1818, 1763 cm⁻¹ (nCO, anhydride, vs); ¹³C {¹H} NMR (THF-d₈): d 171(s, —CHRC(O)O—]. The product was extracted with cold hexanes (0° C., 3×2mL) to yield a red/brown hexanes solution and a pale orange solid.Evaporation of the hexanes solution gave a red oil containing(PPh₃)₂Ni(¹³CO)₂ [¹³C{¹H}NMR (THF-d₈): d 202 (t, Ni(¹³ CO)₂, J_(P-C)=15Hz); IR (THF): 1954, 1895 cm⁻¹ (n¹³CO, vs)], and drying of the solidgave an orange powder which could be purified by precipitation fromTHF/hexanes to give (S)-[NiNHC(H)R¹³C(O)NCH₂R]_(x), R=—CH₂CH(CH₃)₂ (6mg, 80% yield). IR (THF): 3290 cm⁻¹ (nNH, s br), 1536 cm⁻¹ (n¹³CO,amidate, vs). ¹³C {¹H} NMR (THF-d₈): d 182 (s, [NiNHC(H)R¹³C(O)NCH₂R]_(x)).

(S)-(2,2′-bipyridyl)NiNHC(H)RC(O)NCH₂R, R=—CH₂CH₂C(O)OCH₂C₆H₅: 2

In the dry box, a yellow solution of (S)-[NiNHC(H)RC(O)NCH₂R]_(x),R=—CH₂CH₂C(O)OCH₂C₆H₅ (40 mg, 0.085 mmol) in DMF (0.5 mL) was added to asolution of 2,2′-bipyridyl (54 mg, 0.35 mmol) in DMF (0.5 mL). Thehomogeneous mixture was stirred for 2 d at 50° C., during which thecolor changed from yellow to blood red. THF (1 mL) and toluene (5 mL)were layered onto this solution resulting in precipitation of a redpowder. This powder was reprecipitated from DMF/THF/toluene (1:2:10) twoadditional times to give (S)-(2,2′-bipyridyl)NiNHC(H)RC(O)NCH₂R,R=—CH₂CH₂C(O)OCH₂C₆H₅ as a red powder (49 mg, 92% yield). An ¹H NMRspectrum could not be obtained in THF-d₈, most likely because ofparamagnetism of the complex (only broad lines for the benzyl estergroups were observed). IR (THF): 3281 cm⁻¹ (nNH, s br), 1732 cm⁻¹ (nCO,ester, vs), 1597 cm⁻¹ (nCO, amidate, vs). Anal. calcd. for NiC₃₃H₃₄N₄O₅:63.37% C, 5.49% H, 8.95% N. found: 63.72% C, 5.49% H, 8.86% N. [a]_(D)²⁰ (THF, c=0.001)=−135.

Polymerization of Glu-NCA using (S)-(2,2′-bipyridyl)NiNHC(H)RC(O)NCH₂R,R=—CH₂CH₂C(O)OCH₂C₆H₅

In the dry box, Glu NCA (50 mg, 0.2 mmol) was dissolved in DMF (0.5 mL)and placed in a 25 mL reaction tube which could be sealed with a Teflonstopcock. An aliquot of (S)-(2,2′-bipyridyl)NiNHC(H)RC(O)NCH₂R,R=—CH₂CH₂C(O)OCH₂C₆H₅ (50 ml of a 40 mM solution in DMF) was then addedvia syringe to the flask. A stirbar was added and the flask was sealed,removed from the dry box, and stirred in a thermostated 25° C. bath for16 hours. Polymer was isolated by addition of the reaction mixture tomethanol containing HCl (1 mM) causing precipitation of the polymer. Thepolymer was then dissolved in THF and reprecipitated by addition tomethanol. The polymer was dried in vacuo to give a white solid, PBLG (39mg, 93% yield). ¹³C {¹H} NMR, ¹H NMR, and FTIR spectra of this materialwere identical to data found for authentic samples of PBLG. H. Block,Poly(g-benzyl-L-glutamate) and Other Glutamic Acid Containing Polymers,Gordon and Breach, New York, (1983). GPC of the polymer in 0.1M LiBr inDMF at 60° C.: M_(n)=21,600; M_(w)/M_(n)=1.09.

Reaction of (2,2′-bipyridyl)Ni(COD) with ¹³C₂-L-Leucine NCA

In the dry box, five equivalents of ¹³C₂-L-Leucine NCA (14.5 mg, 0.091mmol) was added to a solution of bipyNi(COD) (5.9 mg, 0.018 mmol) in THF(1 mL). The mixture slowly turned from purple to red and was stirred for16 hours. The crude product was isolated by evaporation of the solventto yield a red oily solid. FTIR analysis of the crude reaction mixtureconfirmed the presence of (2,2′-bipyridyl)Ni(CO)₂ (IR (THF): 1978, 1904cm⁻¹ (nCO, vs); Literature: IR (diethyl ether): 1983, 1914 cm⁻¹)],polyleucine [IR (THF): 1653 cm⁻¹ (nAmide I, vs); 1546 cm⁻¹ (nAmide II,vs)] as well as the ¹²C-amidate endgroup [IR(THF): n(CO)=1577 cm⁻¹]. R.S, Nyholm, et al., J. Chem. Soc., 2670 (1953). The reaction was also runin DMF-d₇ (0.5 mL) under otherwise identical conditions. ¹³C {¹H} NMR(DMF-d₇): d 126 (s, ¹³ CO₂).

Reaction of (2,2′-bipyridyl)Ni(COD) with ¹³C₅-L-Leucine NCA

In the dry box, five equivalents of ¹³C₅-L-Leucine NCA (14.5 mg, 0.091mmol) was added to a solution of bipyNi(COD) (5.9 mg, 0.018 mmol) in THF(1 mL). The mixture slowly turned from purple to red and was stirred for16 hours. The crude product was isolated by evaporation of the solventto yield a red oily solid. FTIR analysis of the crude reaction mixtureconfirmed the presence of (2,2′-bipyridyl)Ni(¹³CO)₂ [IR (THF): 1933,1862 cm⁻¹ (nCO, vs)] as well as ¹³C-labeled polyleucine [IR (THF): 1613cm⁻¹ (nAmide 1, vs); 1537 cm⁻¹ (nAmide II, vs)]. The reaction was alsorun in DMF-d₇ (0.5 mL) under otherwise identical conditions. ¹³C {¹H}NMR (DMF-d₇): d 198 (s, bipyNi(¹³ CO)₂); 177 (s,bipyNiN(H)C(H)R¹³C(O)N[CH(R)¹³ C(O)NH]_(n)CH₂R), 174 (s,bipyNiN(H)C(H)—R¹³ C(O)N[CH(R)¹³C(O)NH]_(n)CH₂R).

Polymerization of Glu-NCA with (2,2′-bipyridyl)Ni(COD)

In the dry box, Glu NCA (50 mg, 0.2 mmol) was dissolved in DMF (0.5 mL)and placed in a 25 mL reaction tube which could be sealed with a Teflonstopcock. An aliquot of bipyNi(COD) (50 ml of a 40 mM solution in DMF)was then added via syringe to the flask. A stirbar was added and theflask was sealed, removed from the dry box, and stirred in athermostated 25° C. bath for 16 hours. Polymer was isolated by additionof the reaction mixture to methanol containing HCl (1 mM) causingprecipitation of the polymer. The polymer was then dissolved in THF andreprecipitated by addition to methanol. The polymer was dried in vacuoto give a white solid, PBLG (41 mg, 98% yield). ¹³C {¹H} NMR, ¹H NMR,and FTIR spectra of this material were identical to data found forauthentic samples of PBLG. H. Block, Poly(g-benzyl-L-glutamate) andOther Glutamic Acid Containing Polymers, Gordon and Breach, New York,(1983). GPC of the polymer in 0.1M LiBr in DMF at 60° C.: M_(n)=22,100;M_(w)/M_(n)=1.15.

As discussed above, a-Amino acid-N-carboxyanhydrides (NCAs) were reactedwith zerovalent nickel complexes of the type L₂Ni(COD) to yieldmetallacyclic oxidative addition products. These oxidative additionreactions were found to result in the addition across either the O—C₅ orthe O—C₂ bond of the NCAs, ultimately giving, after addition of a secondequivalent of NCA, chiral amido-amidate nickelacycles. The origins andstructures of these complexes were elucidated by use of selectively ¹³Clabeled NCA reagents. Stable metallacycles were obtained when L=PPh₃.When other donor ligands were used, the metallacycle intermediates werefound to quickly react with additional NCA molecules to formpolypeptides in quantitative yield and with narrow molecular weightdistributions. These reactions provide a facile route to unusuallystable metallacyclic amido-containing nickel intermediates.

When two equivalents of PPh₃ and one Ni(COD)₂ were reacted with oneequivalent of g-benzyl-L-glutamate-N-carboxyanydride (Glu-NCA) in THF atroom temperature, rapid consumption of the NCA was observed. From thegolden brown solution, an alkane soluble brown oil and a THF solubleyellow powder were isolated. Analysis of the oil confirmed the presenceof (PPh₃)₂Ni(CO)₂ [IR(THF): n(CO)=2000, 1939 cm⁻¹] which was produced bythe decarbonylation of an intermediate six-membered metallacyclefollowed by trapping of the carbon monoxide with (PPh₃)₂Ni(COD). H.Kanazawa, et al., Bull. Chem. Soc. Jpn., 51:2205-2208 (1978). Infraredanalysis of the yellow powder showed carbonyl stretches at 1734 and 1577cm⁻¹ which were assigned, respectively, to the side-chain benzyl estersand amidate group of the chiral nickelacycle (see scheme 2 of FIG. 3).

The structures and origins of these products were elucidated when¹³C₅-L-leucine-N-carboxyanhydride was reacted with (PPh₃)₂Ni(COD) inTHF. An infrared spectrum of the crude reaction mixture showed a stretchat 1536 cm⁻¹ for the ¹³C-amidate group [¹³C {¹H} NMR (THF-d₈): 182 ppm]as well as (PPh₃)₂Ni(¹³CO)₂ stretches at 1954 and 1895 cm⁻¹ (¹³C {¹H}NMR (THF-d₈): 202 ppm] which were isotopically shifted from theunlabeled compounds (see equation 1 of FIG. 4). When¹³C₂-L-leucine-N-carboxyanhydride was reacted with (PPh₃)₂Ni(COD) inTHF, analysis of the products showed exclusive formation of(PPh₃)₂Ni(¹²CO)₂ [IR(THF): n(CO)=2000, 1939 cm⁻¹] and the ¹²C amidate[IR(THF): n(CO)=1580 cm⁻¹] (see equation 2 of FIG. 4). Since no mixed¹³C/¹²C products were observed, it was concluded that oxidative additionwas occurring either at the C₅—O or the C₂—O bond followed bydecarbonylation and addition of a second NCA molecule to yield anamido-containing nickelacycle (see scheme 2 of FIG. 3).

While not being bound to a specific mechanism or theory, thetransformation of the initially formed 6-membered amido-alkylnickelacycle to a 5-membered amido-amidate nickelacycle might occurthrough a proton-transfer mediated ring contraction induced by additionof an additional NCA monomer (see equation FIG. 4). Such atransformation has been observed for related nickel complexes.

Furthermore, quenching of polymerization reactions with DCl and HCl hasshown conclusively that the nickel-alkyl bond of the 6-memberedmetallacycle is not present after addition of NCA molecules to theinitial metallacycle, providing additional supporting evidence for thering-contraction to the amido-amidate structure.

The structure of these metallacyclic products was further confirmed byelemental analysis and acidolysis of the complexes. The productmetallacycles contain no phosphine by elemental analysis and were foundto consist of the empirical formula [NiNHC(H)RC(O)NCH₂CHR]_(x). Osmoticmolecular weight measurements in THF (ca. 7 mg/mL) showed that thecomplexes aggregate as dimers. Treatment of the metallacyclic complexderived from L-leucine NCA with HCl in THF gave only a single organicproduct. Analysis of this product by ¹H NMR spectroscopy andpolarimetry, and comparison of the data with an authentic sample, showedit to be optically pure L-leucine isoamylamide.HCl (see scheme 2 of FIG.3).

When the donor ligands bound to the nickel (0) precursor were varied(e.g. alkyl phosphines, a,a′-diimines), the only products isolable fromstoichiometric reactions with Glu-NCA in THF were some startingnickel(0) compound and poly(g-benzyl-L-glutamate), PBLG. When 100equivalents of Glu-NCA was added to bipyNi(COD) in DMF, all of thenickel precursor was consumed and PBLG was isolated in excellent yield(>95%) with narrow molecular weight distribution (M_(n)=22,100,M_(w)/M_(n)=1.15). L₂=donor ligand(s); COD=1,5-cyclooctadiene;bipy=2,2′-bipyridyl. It has been shown that bipyNi(COD) initiates theliving polymerization of NCAs. T. J. Deming, Nature, 390:386-389 (1997).It was suspected that bipyNi(COD) oxidatively adds Glu-NCA to form theactive polymerization initiator in situ which then rapidly consumes theremainder of the monomer. To identify this active initiator, a series ofexperiments were performed where bipyNi(COD) was reacted withselectively ¹³C labeled NCA monomers.

In order to completely consume all the bipyNi(COD) in reactions withNCAs, at least a fivefold excess of NCA monomer was used. BipyNi(COD)was reacted with five equivalents of ¹³C₅-L-leucine-N-carboxyanhydridein THF. IR and ¹³C {¹H} NMR analysis of the crude products verified thepresence of bipyNi(¹³CO)₂ [IR(THF): n(¹³CO)=1933, 1862 cm⁻¹; ¹³C {¹H}NMR (DMF-d₇): 198 ppm], ¹³C-labeled poly-L-leucine [IR (THF): 1613 cm⁻¹(nAmide 1, vs); 1537 cm⁻¹ (nAmide II, vs); ¹³C {¹H} NMR (DMF-d₇): 177ppm (bipyNiN(H)C(H)R¹³C(O)N[CH(R)¹³ C(O)—NH]_(n)CH₂R)], and the labelednickel-amidate endgroup [¹³C {¹H} NMR (DMF-d₇): 174 ppm(bipyNiN(H)C(H)R¹³ C(O)N—[CH(R)¹³C(O)NH]_(n)CH₂R)]. The reaction with¹³C₂-L-leucine-N-carboxyanhydride gave similar products, except forlocation of the ¹³C label. The presence of bipyNi(¹²CO)₂ [IR(THF):n(CO)=1978, 1904 cm⁻¹],¹² poly-L-leucine [IR (THF): 1653 cm⁻¹ (nAmide 1,vs); 1546 cm⁻¹ (nAmide II, vs)],¹³ as well as the ¹²C-amidate endgroup(IR(THF): n(CO)=1577 cm⁻¹] was identified. When the reaction was run inDMF-d₇, the presence of liberated ¹³CO₂ was also confirmed using ¹³C{¹H} NMR [126 ppm (s, ¹³ CO₂)].

All of these experiments were consistent with initial addition of theNCA to bipyNi(COD) across the C₅—O bond, analogous to the reactionsusing (PPh₃)₂Ni(COD). The primary influence of the ligands manifestsitself in the reactivity of the resulting products. The ligand freecomplex from the PPh₃ reaction was inert toward further reactivity withNCAs, while the bipy complex and complexes formed with othera,a′-diimines and alkyl phosphines were efficient NCA polymerizationinitiators. This phenomenon was directly verified by synthesis of thereactive metallacycle intermediate formed in the bipyNi(COD)/NCAreactions. The stable metallacycle (S)-[NiNHC(H)RC(O)NCH₂R]_(x),R=—CH₂CH₂C(O)OCH₂C₆H₅) was reacted with an excess of bipy in DMF to formthe ligand adduct (S)-(2,2′-bipyridyl)NiNHC(H)RC(O)NCH₂R,R=—CH₂CH₂C(O)OCH₂C₆H₅ (see scheme 2 of FIG. 3). Reaction of(S)-(2,2′-bipyridyl)NiNHC(H)RC(O)NCH₂R, R=—CH₂CH₂C(O)OCH₂C₆H₅ with 100equivalents of Glu-NCA in DMF resulted in rapid polymer formation. ThePBLG formed in this reaction was identical to that formed usingbipyNi(COD) under otherwise identical conditions (M_(n)=21,600,M_(w)/M_(n)=1.09). The bipyNi(COD) mediated polymerizations of NCAs aretherefore thought to proceed via amido-amidate nickelacycle activeendgroups (see equation 4 of FIG. 4).

Example 2 Initiators for Chain-End Functionalized Polypeptides and BlockCopolypeptides

N_(α)-allyloxycarbonyl-amino acid amides were reacted with zerovalentnickel complexes LNi(1,5-cyclooctadiene) (L=2,2′-bipyridine (bpy),1,10-phenanthroline (phen), 1,2-bis(dimethylphosphino)ethane (dmpe), and1,2-bis(diethylphosphino)ethane (depe)), to yield amido-amidatemetallacycles of the general formula: LNiNHC(R′)HC(O)NR″. Thesecomplexes were found to initiate polymerization of α-aminoacid-N-carboxyanhydrides (NCAs) yielding polypeptides with definedmolecular weights, narrow molecular weight distributions, and withquantitative incorporation of the initiating ligand as an end-group. Anapthyl substituent was incorporated as a fluorescent end-group todemonstrate the feasibility of this methodology.

General Experimental Protocols and Reagents

Infrared spectra were recorded on a Perkin Elmer 1605 FTIRSpectrophotometer calibrated using polystyrene film. Tandem gelpermeation chromatography/light scattering (GPC/LS) was performed on anSSI Accuflow Series III liquid chromatograph pump equipped with a WyattDAWN DSP light scattering detector and Wyatt Optilab DSP. Separationswere effected by 10⁵ Å, 10³ Å, and 500 Å Phenomenex 5μ columns using0.1M LiBr in DMF eluent at 60° C. NMR spectra were measured on BrukerAVANCE 200 MHz spectrometer. FAB Mass Spectrometry was performed at thefacility in the Chemistry Department at the University of California,Santa Barbara. MALDI mass spectra were collected using a ThermoBioAnalysis DYNAMO mass spectrometer running in positive ion mode withsamples prepared by mixing solutions of analyte in TFA with solutions of2,5-dihydroxybenzoic acid in TFA and allowing the mixtures to air dry.Fluorescence measurements were conducted on a SPEX FluoroMax.-2.Chemicals were obtained from commercial suppliers and used withoutpurification unless otherwise stated. Alloc-L-amino amides,ε-CBZ-L-lysine NCA, (S)-phenylglycine NCA, and γ-benzyl-L-glutamate NCAwere prepared according to literature procedures (Tirrell, J. G.;Fournier, M. J.; Mason, T. L.; Tirrell, D. A. Chem. & Engr. News 1994,72, 40-51; Viney, C.; Case, S. T.; Waite, J. H. Biomolecular Materials,Mater. Res. Soc. Proc. 292, 1992.; and Deming, T. J. J. Am. Chem. Soc.,1998, 120, 4240-4241: incorporated herein by reference). Hexanes, THF,and THF-d₈ were purified by first purging with dry nitrogen, followed bypassage through columns of activated alumina ³DMF and DMF-d₇ werepurified by drying over 4 Å molecular sieves followed by vacuumdistillation.

Sample Procedure for Synthesis of Alloc-L-Amino Acid Amides: Alloc-LLeucine-Isoamylamide

Isoamylamine (1.4 mL, 12 mmol) was added to a solution ofAlloc-L-leucine-N-hydroxysuccinimidyl ester (2.5 g, 8.0 mmol) in THF (5mL). The reaction was stirred for 1 hr after which the resultingprecipitate was removed by filtration and the solution was diluted withethyl acetate (100 mL). This solution was sequentially washed withdilute aqueous HCl (2×30 mL), saturated aqueous NaHCO₃ (2×30 mL), andthen saturated aqueous NaCl (2×30 mL) followed by drying over MgSO₄. Thesolvent was then evaporated in vacuo to leave the product (1.7 g, 77%).IR(THF): 1724 cm⁻¹ (υCO, Alloc, s), 1674 cm⁻¹ (υCO, amide, s). ¹H NMR(CDCl₃): δ 6.00 (br s,(CH₃)₂—CHCH₂CH(NHC(O)OCH₂CH═CH₂)C(O)NHCH₂CH₂CH(CH₃)₂, 2H), 5.85 (m,(CH₃)₂—CHCH₂CH(NHC(O)OCH₂CH═CH₂)C(O)NHCH₂CH₂CH(CH₃)₂, 1H), 5.25 (t,(CH₃)₂—CHCH₂CH(NHC(O)OCH₂CH═CH ₂)C(O)NHCH₂CH₂CH(CH₃)₂, 2H), 4.57 (d,CH₃CHCH₂CH(NHC(O)OCH ₂CH═CH₂)C(O)NHCH₂CH₂CH(CH₃)₂, 2H), 4.12 (m,(CH₃)₂CHCH₂CH(NHC(O)OCH₂CH═CH₂)C(O)NHCH₂CH₂CH(CH₃)₂, 1H), 3.24 (q,(CH₃)₂CHCH₂CH(NHC(O)OCH₂CH═CH₂)C(O)NHCH ₂CH₂CH(CH₃)₂, 2H), 1.57 (m,(CH₃)₂CHCH ₂CH(NHC(O)OCH₂CH═CH₂)C(O)NHCH₂CH₂CH(CH₃)₂, 2H), 1.40 (m,(CH₃)₂CHCH ₂CH(NHC(O)OCH₂CH═CH₂)C(O)NHCH₂CH ₂CH(CH₃)₂, 4H), 0.92 (d, (CH₃)₂CHCH₂CH(NHC(O)OCH₂CH═CH₂)C(O)NHCH₂CH₂CH(CH ₃)₂, 12H).

(S)-phenNiNHC(H)RC(O)O, R=—CH₂CH(CH₃)₂

1,10-Phenanthroline (phen) (13 mg, 0.073 mmol) was added to a suspensionof Ni(COD)₂ (20 mg, 0.073 mmol) in DMF (2 mL and let stand at roomtemperature for 30 min after which a solution of Phen-Ni(COD) hadformed. Alloc-L-leucine allyl ester (20 mg, 0.073 mmol) was added to thepurple solution, which subsequently became brown in color. Afterstanding at room temperature for 5 h the solution was green, indicativeof formation of the single oxidative-addition product. The greensolution was heated at 80° C. for 20 h to yield a purple solution. Theproduct was isolated from this solution by precipitation into diethylether (10 mL) followed by washing with THF (2×10 mL) and drying-, invacuo to give a purple powder (16 mg, 68%). IR(THF): 1620 cm⁻¹ (υCO,carboxylate, s br). An ¹H NMR spectrum could not be obtained in DMF-d,most likely because of paramagnetism of the complex (only broad linesfor the methyl groups were observed). μ_(eff) (296K)=2.05, μ_(B).

(S)-phenNiNHC(H)RC(O)NCH₂R, R=—CH₂CH(CH₃)₂, 2

Phen (13 mg, 0.073 mmol) was added to a suspension of Ni(COD)₂ (20 mg,0.073 mmol) in DMF (2 mL) and let stand at room temperature for 30 minafter which a solution of phenNi(COD) had formed. Alloc-L-leucineisoamyl amide (20 mg, 0.073 mmol) was then added to the purple solution,which subsequently became brown in color. After standing at roomtemperature for 5 h the solution was green, indicative of formation ofthe single oxidative-addition product. The green solution was heated at80° C. for 20 h to yield a purple solution. The product was isolatedfrom this solution by precipitation into diethyl ether (10 mL) followedby washing with THF (2×10 mL) and drying in vacuo to give a purplepowder (23 mg, 75%). IR(THF): 1578 cm−1 (υCO, amidate, s br). An ¹H NMRspectrum could not be obtained in DMF-d, most likely because ofparamagnetism of the complex (only broad lines for the methyl groupswere observed). μ_(eff) (296K)=2.34 μ_(B).

(S)-phenNiNHC(H)R¹³C(O)NCH₂R, R=—CH₂CH(CH₃)₂, 2-¹³C

Phen (13 mg, 0.073 mmol) was added to a suspension of Ni(COD)₂ (20 mg,0.073 mmol) in DMF (2 mL) and let stand at room temperature for 30 minafter which a solution of phenNi(COD) had formed.¹³C(amide)-alloc-L-leucine isoamyl amide (20 mg, 0.073 mmol) was thenadded to the purple solution, which subsequently became brown in color.After standing at room temperature for 5 h the solution was green,indicative of formation of the single oxidative-addition product. Thegreen solution was heated at 80° C. for 20 h to yield a purple solution.The product was isolated from this solution by precipitation intodiethyl ether (10 mL) followed by washing with THF (2×10 mL) and dryingin vacuo to give a purple powder (22 mg, 70%) IR(THF): 1541 cm⁻¹ (υ¹³CO,amidate, s br).

(S)-depeNiNHC(H)R¹C(O)NCH₂R²—, R¹=—CH₂(CH₃)₂, R²=—CH₂CH₃, 4

1,2-Bis(diethylphosphino)ethane, depe (17 μL, 0.073 mmol) was added to asolution of Ni(COD)₂ (20 mg, 0.073 mmol) in THF (1 mL) and let stand atroom temperature for 5 min after which a solution of depeNi(COD) hadformed. Alloc-L-valine n-propyl amide (18.5 mg, 0.073 mmol) in DMF (1mL) was then added to the yellow solution, which subsequently becameorange-yellow in color. The solution was heated at 80° C. for 20 h toyield an orange solution. The solvent was removed in vacuo and theresidue was redissolved in THF and isolated from this solution byprecipitation into hexanes (10 mL). Drying of the solid in vacuo gave 4as a yellow powder (16 mg, 53%). IR(THF): 1578 cm⁻¹ (υCO, amidate, sbr). An ¹H NMR spectrum could not be obtained in DMF-d, most likelybecause of paramagnetism of the complex (only broad lines for the alkylgroups were observed). μ_(eff) (296K)=2.08 μ_(B).

Isolation of L-Leucine-HCl from (S)-phenNiNHC(H)RC(O)O, R═CH₂CH(CH₃)₂

Anhydrous 4M HCl in dioxane solution (1.0 mL) was added to a solution of(S)-PhenNiNHC(H)RC(O)O, R=—CH₂CH(CH₃)₂ (10 mg, 0.027 mmol) in CH₂Cl₂.The solution immediately changed color from purple to orange. It wasstirred for 2 hours and then the solvents were removed in vacuo. Theremaining solid was extracted with water and the insolublenickel-containing residue was removed by filtration. The water was thenremoved by freeze-drying to yield the desired product. FAB-MS: M-Cl⁻:132.19 calcd, 132 found.

Isolation of L-Leucine Allylamide-HCl from (S)-bpyNiNHC(H)R¹C(O)NR²,R²=—CH₂CH(CH₃ 2, R²=—CH₂CH═CH₂, 1

Anhydrous 4M HCl in dioxane solution (1.0 mL) was added to a solution of1 (10 mg, 0.025 mmol) in CH₂Cl₂. The solution immediately changed colorfrom purple to orange. It was stirred for 2 hours and then the solventswere removed in vacuo. The remaining solid was extracted with water andthe insoluble nickel-containing residue was removed by filtration. Thewater was then removed by freeze-drying to yield the desired product. ¹HNMR (D₂O): δ 5.91 (m, (CH₃)₂CHCH₂CH(NH₂)C(O)—NHCH₂CH═CH₂, 1H), 5.73 (t,(CH₃)₂CHCH₂CH(NH₂)—C(O)NHCH₂CH═CH, 2H), 3.95 (m,(CH₃)₂CHCH₂CH(NH₂)C(O)NHCH₂CH═CH₂, 1H), 3.81 (br s,(CH₃)₂CHCH₂CH(NH₂)—C(O)NHCH ₂CH═CH₂, 2H), 1.74 (d, (CH₃)₂CHCH₂CH(NH₂)C(O)NHCH₂CH═CH₂ 3H), 0.96 (d, (CH₃)₂CHCH₂CH(NH₂)C(O)NHCH₂CH═CH₂, 6H). FAB-MS: M-Cl⁻: 171.28 calcd, 171found.

Isolation of L-Leucine Isoamylamide-HCl from(S)-phenNiNHC(H)RC(O)—NCH₂R, R=—CH₂CH(CH₃)₂, 2

Anhydrous 4M HCl in dioxane solution (1.0 m-L) was added to a solutionof 2 (10 mg, 0.024 mmol) in CH₂Cl₂. The solution immediately changedcolor from purple to orange. It was stirred for 2 hours and then thesolvents were removed in vacuo. The remaining solid was extracted withwater and the insoluble nickel-containing residue was removed byfiltration. The water was then removed by freeze-drying to yield thedesired product. ¹H NMR (D₂O): δ 3.94 (t, NH₃CH(CH₂CH(CH₃)₂)C(O)—, 1H,J=7.5 Hz), 3.33, 3.14 (dm, —C(O)NHCH ₂CH₂CH(CH₃)₂, 2H, J_(gem)=10.7 Hz,J_(mult)=6 Hz, 13 Hz), 1.72 (dd, NH₃CH(CH ₂CH(CH₃)₂C(O)—, 2H, J=6 Hz, 7Hz), 1.68 (m, NH₃CH—(CH₂CH(CH₃)₂)C(O)—, 1H, J=7 Hz), 1.63 (m,—C(O)NHCH₂CH₂CH(CH₃)₂, 1H, J=7 Hz), 1.43 (ddd, —C(O)NHCH₂CH₂CH(CH₃)₂,2H, J=7 Hz), 0.98 (d, NH₃CH(CH₂CH(CH ₃)₂)—C(O)—, 3H, J=6 Hz), 0.96 (d,NH₃CH(CH₂CH(CH₃)₂)C(O)—, 3H, J=6 Hz), 0.92 (d, C(O)—NHCH₂CH₂CH(CH ₃)₂,3H, J=6 Hz), 0.90 (d, —C(O)NHCH₂CH₂CH—(CH ₃)₂, 3H, J=6 Hz). FAB-MS:M-Cl⁻: 201.36 calcd, 201 found.

Polymerization of Glu NCA Using (S)-depeNiNHC(H)R¹C(O)NR²,R¹=—CH₂CH(CH₃)₂, R²=-1-Naphthyl, 3

In the dry box, γ-benzyl-L-glutamate-N-carboxyanhydride, Glu NCA (50 mg,0.2 mmol) was dissolved in DMF (1.0 mL) and placed in a 25 mL reactiontube which could be sealed with a Teflon stopcock. An aliquot of 3 (140μL of a 14 mM solution in DMF) was added via syringe to the flask. Astir bar was added and the flask was sealed, removed from the dry boxand stirred at 25° C. in a thermostated bath for 24 h. Polymer wasisolated by addition of the reaction mixture to methanol containing HC1(1 mM) causing precipitation of the polymer. The polymer was dried invacuo to give a white solid, PBLG (19 mg, 90% yield) ¹³C{¹H} NMR, ¹HNMR, and FTIR spectra of this material were identical to data found forauthentic samples of PBLG.⁴ GPC of the polymer in 0.1 M LiBr in DMF at60° C.: M_(n)=26,100; M_(w)/M_(n)=1.15.

Isolation of Mixed Hexenes from Oxidative Addition of N_(α)-trans2-Hexenyloxycarbonyl-L-Leucine Isoamyl Amide to Nickel

A solution of trans 2hexenyloxycarbonyl-L-leucine isoamyl amide (231 mg,0.015 mmol) in THF (0.5 mL) was added to a solution of (PPh₃)₄Ni (741mg, 0.015 mmol) in THF (0.5 mL) resulting in a yellow-orange solution.This mixture was heated at 80° C. for 2 days during which the colorchanged to dark brown. The volatiles of the reaction were then vacuumdistilled into an NMR tube to remove the paramagnetic nickel products.The presence of a mixture of 1-hexene, 2-hexenes and 3-hexenes in thedistillate was verified by ¹³C NMR. The mixture of hexenes was likelyformed by facile isomerization of the intermediate η³hexenyl-nickelspecies formed in the reaction. ¹³C NMR (THF): δ 139.05(CH₃CH₂CH₂CH₂CH═CH₂), 132.15 (trans-CH₃CH₂ CH═CHCH₂CH₃), 131.44(CH₃CH₂CH₂CH═CHCH₃), 130.53 (trans-CH₃CH₂CH₂CH═CHCH₃), 128.56(cis-CH₃CH₂ CH═CHCH₂CH₃), 125.92 (cis-CH₃CH₂CH₂ CH═CHCH₃), 124.79(trans-CH₃CH₂CH₂ CH═CHCH₃), 114.10 (CH₃CH₂CH₂CH₂ CH═CH₂), 34.95(trans-CH₃CH₂ CH₂CH═CHCH₃), 33.73 (CH₃CH₂ CH₂CH₂CH═CH₂), 31.44(CH₃CH₂CH₂ CH₂CH═CH₂), 29.71 (cis-CH₃CH₂ CH₂CH═CHCH₃), 22.89 (CH₃CH₂CH₂CH₂CH═CH₂), 22.38 (trans-CH₃CH₂CH₂CH═CHCH₃), 18.05 (trans-CH₃CH₂CH₂CH═CHCH₃), 15.04 (cis-CH₃CH₂CH₂CH═CHCH₃), 14.44 (trans-CH₃CH₂CH═CHCH₂CH₃), 13.79 (cis-CH₃ CH₂CH₂CH═CHCH₃), 13.60(CH₃CH₂CH₂CH₂CH═CH₂), 13.32 (trans-CH₃CH₂CH₂CH═CHCH₃), 13.20(cis-CH₃CH₂CH₂CH═CHCH₃).

Polymerization of Glu NCA with (S)-PhenNiNHC(H)RC(O)O, R=—CH₂CH(CH₃)₂

In the dry box, Glu NCA (50 mg, 0.2 mmol) was dissolved in DMF (1.0 mL)and placed in a 25 mL reaction tube which could be sealed with a Teflonstopcock. An aliquot of the initiator (100 μL of a 36 mM solution inDMF) was added via syringe to the flask. A stir bar was added and theflask was sealed, removed from the dry box and stirred at 25° C. in athermostated bath for 24 h. Polymer was isolated by addition of thereaction mixture to methanol containing HCl (1 mM) causing precipitationof the polymer. The polymer was dried in vacuo to give a white solid,PBLG (18.1 mg, 87% yield) ¹³C{¹H} NMR, ¹H NMR, and FTIR spectra of thismaterial were identical to data found for authentic samples of PBLG(Stevens, C.; Watanabe, R. J. Am. Chem. Soc., 1950, 72, 725-727). GPC ofthe polymer in 0.1 M LiBr in DMF at 60° C.: M_(n)=45,500;M_(w)/M_(n)=1.24.

Fluorescence Measurements of 1-Napthyl Functionalized PBLG

A solution of 1-napthyl functionalized PBLG (26.5 mg) in THF (2 ml) wasplaced into a cuvette. The sample was excited at a frequency of 324 nmwhich yielded an emission with maximum intensity at 390 nm. Thisemission was characteristic of the 1-napthyl end-group. When themolecular weights of the polymers were varied, the corresponding,emission intensities were found to vary inversely with chain length,indicating that the number of end-groups was proportional to the numberof chains. Control experiments showed that the emission from labeledpolymers was an order of magnitude greater than that from unlabeled PBLG(see Table 3 below).

TABLE 3 Entry Polymer Mass(mg) M_(n) Intensity(cps) I PBLG-Nap 26.514400 7593550 2 PBLG-Nap 26.5 26100 4238040 3 PBLG 26.5 36100 800000

Isolation of cis-5-Norbornene-endo-2-Carboxylic acid-3-Carboxyl L-Valinen-Propyl Amide from Reaction of 4

A solution of cis-5-norbornene-endo-2,3-dicarboxylic anhydride (6.0 mg,0.037 mmol) in THF (1 mL) was added to a solution of 4 (16 mg, 0.037mmol) in THF (1 ml). The yellow solution was heated at 40° C. for 2 duntil the anhydride stretch at 1780 cm¹ was no longer detectable byFTIR. A dilute solution of HCl in water (0.5 mL) was added to thereaction which then immediately changed color from yellow to orange. Themixture was stirred for 2 h and then the volatiles were removed invacuo. The remaining-solid was extracted with THF, filtered, and thenadded to Et₂O to precipitate the nickel-containing byproducts. Thesolubles were then condensed in vacuo to yield the product (11 mg, 92%).FAB-MS: MH⁺: 323.8 calc, 323 found.

Polymerization of (S)-phenylglycine NCA Using(S)-depeNiNHC(H)R¹—C(O)NR², R¹=—CH₂CH(CH₃)₂, R²=—CH₂CH₂CH(CH₃)₂, 7

In the dry box, (S)-phenylglycine NCA (50 mg, 0.28 mmol) was dissolvedin THF (1.0 mL) and placed in a 25 mL reaction tube which could besealed with a Teflon stopcock. An aliquot of 7 (560 μL of a 50 mMsolution in THF) was added via syringe to the flask. A stir bar wasadded and the flask was sealed, removed from the dry box and stirred at25° C. in a thermostated bath for 24 h. Polymer was observed toprecipitate from solution during this time period. Polymer was isolatedby addition of the reaction mixture to methanol containing HCl (1 mM)followed by centrifugation. The polymer was washed with excess water,methanol, and then diethyl ether and then dried in vacuo to give theproduct as a white solid (35 mg, 93% yield). ¹H NMR (TFA-d) and FTIRspectra of this material were identical to data found for authenticsamples of poly (S)-phenylglycine. MALDI mass spectroscopy of thepolymer showed a distribution of masses ranging from ca. 1000-4500 Da,with the separation between the peaks equal to the mass of thephenylglycine repeats (133.15 Da). Below 1000 Da, the spectra werecomplicated by the presence of large amounts of matrix peaks. Analysisof the absolute masses of the peaks revealed that nearly all chains wereend-functionalized with the leucine residue of the initiator (FIG. 7).Some of the chains contained the intact leucine isoamylamide end-group(b-series), while the remainder contained a leucine end-group where theC-terminal amide had been cleaved by hydrolysis after dissolution in wetTFA (a-series). As an example peak, 9a: expected MH⁺: 1331.44 Da; foundMH⁺: 1330.13 Da. Only very small peaks were observed wherenon-functionalized oligo(phenylglycines) should appear (c-series), andthese peaks may also contain adducts formed with functionalized chains.For example, 10c (1350.43 Da) has a mass nearly equal to 9a+O (1347.44Da) [peak observed at 1347.53 Da]. From comparison of the peakintensities for the a- and b-series of peaks (and adducts) to thec-series of peaks, it was determined that the degree of chainfunctionalization was greater than 98%.

The initiators described above were generated usingbis-1,5-cyclooctadiene nickel (Ni(COD)₂) as the nickel source inconjunction with a variety of donor ligand components. We have alsosuccessfully used other sources of zerovalent nickel (e.g. Ni(CO)₄) aswell as other donor ligands (e.g., PR₃ [R=Me, Et, Bu, cyclohexyl,phenyl], R₂PCH₂CH₂PR₂ [R=Me, phenyl], (α,α′-diimine ligands[1,10-phenanthroline, neocuproine], diamine ligands [tetramethylethylenediamine], and isocyanide ligands [tert-butyl isocyanide]) to generatethese initiators. The use of other sources of zerovalent nickel (e.g.nickel-olefin complexes, nickel-carbonyl complexes, nickel-isocyanide orcyanide complexes, and nickel nitrogen or phosphorus donor ligandcomplexes) are possible modifications which should not be interpreted asgoing beyond the concept of this invention. Likewise, the use of otherdonor ligands (nitrogen or phosphorus based in particular) or reactionsolvents are logical extensions of this work. Finally, we have foundthat other transition metals, specifically palladium, platinum, cobalt,rhodium and iridium, might also be used in the reaction with alloc-aminoacid amides to form amido-amidate metallacycle initiators, and are thusadditional potential modifications to this invention.

Preparation of Cysteineamide-Polyglutamate-b-Polyleucine block copolymerusing (S)-depeNiNHC(H)R¹C(O)NR², R¹=—CH₂SSC(CH₃)₃, R²=—CH₂C(CH₃)₃, 11

In the dry box, gamma-benzyl-L-glutamate-N-carboxyanhydride, Glu-NCA(280 mg, 1.06 mmol) was dissolved in DMF (2.0 mL) and placed in a 25 mLreaction tube which could be sealed with a Teflon stopcock. An aliquotof 11 (200 mL of a 36 mM solution in DMF) was added via syringe to theflask. A stir bar was added and the flask was sealed, removed from thedry box and stirred at 25° C. in a thermostated bath for 4 h.L-Leucine-N-carboxyanhydride, Leu-NCA (200 mg, 1.26 mmol) was dissolvedin DMF (2.0 mL) and added to the solution in a 25 mL reaction tube thatcould be sealed with a Teflon stopcock. The flask was sealed, removedfrom the dry box and stirred at 25° C. in a thermostated bath for 20 h.The polymer was isolated by addition of the reaction mixture to methanolcontaining HCl (1 mM) causing precipitation of the polymer. The samplewas dried in vacuo to give the block copolypeptide as a white solid (329mg, 87% yield).

Example 3 Facile Synthesis of Block Copolypeptides of DefinedArchitecture

General Experimental Protocols and Reagents

Infrared spectra were recorded on a Perkin Elmer 1605 FTIRSpectrophotometer calibrated using polystyrene film. Tandem gelpermeation chromatography/light scattering (GPC/LS) was performed on aSpectra Physics Isochrom liquid chromatograph pump equipped with a WyattDAWN DSP light scattering detector and Wyatt Optilab DSP. Separationswere effected by 10⁵ Å and 10³ Å Phenomenex 5μ columns using 0.1M LiBrin DMF at 60° C. as eluent. Optical rotations were measured on a PerkinElmer Model 141 Polarimeter using a 1 mL volume cell (1 dm length). NMRspectra were measured on a Bruker AMX 500 MHz spectrometer. Chemicalswere obtained from commercial suppliers and used without purificationunless otherwise stated. (COD)₂Ni was obtained from Strem Chemical Co.,and ¹³C₁-L-leucine and ¹³C-phosgene were obtained from Cambridge IsotopeLabs. g-Benzyl-L-glutamate NCA were prepared according to literatureprocedures. Hexanes, THF, and THF-d₈ were purified by distillation fromsodium benzophenone ketyl. DMF and DMF-d₇ were purified by drying over 4Å molecular sieves followed by vacuum distillation.

Reaction of (2,2′-bipyridyl)Ni(COD) with ¹³C₂-L-Leucine NCA

In the dry box, five equivalents of ¹³C₂-L-Leucine NCA (14.5 mg, 0.091mmol) was added to a solution of bipyNi(COD) (5.9 mg, 0.018 mmol) in THF(1 ml). The mixture slowly turned from purple to red and was let stirfor 16 hours. The crude product was isolated by evaporation of thesolvent to yield a red oily solid. FTIR analysis of the crude reactionmixture confirmed the presence of (2,2′-bipyridyl)Ni(CO)₂ [IR (THF):1978, 1904 cm⁻¹ (nCO, vs), polyleucine [IR (THF): 1653 cm⁻¹ (nAmide 1,vs); 1546 cm⁻¹ (nAmide II, vs)] as well as the ¹²C-amidate endgroup[IR(THF): n(CO)=1577 cm⁻¹]. The reaction was also run in DMF-d₇ (0.5 mL)under otherwise identical conditions. ¹³C {¹H} NMR (DMF-d₇): d 126 (s,¹³ CO₂).

Reaction of (2,2′-bipyridyl)Ni(COD) with ¹³C₅-L-Leucine NCA

In the dry box, five equivalents of C₅-L-Leucine NCA (14.5 mg, 0.091mmol) was added to a solution of bipyNi(COD) (5.9 mg, 0.018 mmol) in THF(1 ml). The mixture slowly turned from purple to red and was let stirfor 16 hours. The crude product was isolated by evaporation of thesolvent to yield a red oily solid. FTIR analysis of the crude reactionmixture confirmed the presence of (2,2′-bipyridyl)Ni(¹³CO)₂ [IR (THF):1933, 1862 cm⁻¹ (nCO, vs)] as well as ¹³C-labeled polyleucine [IR (THF):1613 cm⁻¹ (nAmide 1, vs); 1537 cm⁻¹ (nAmide II, vs)]. The reaction wasalso run in DMF-d₇ (0.5 mL) under otherwise identical conditions. ¹³C{¹H} NMR (DMF-d₇): d 198 (s, bipyNi(¹³CO)₂); 177 (s,bipyNiN(H)C(H)R¹³C(O)N[CH(R)¹³ C(O)—NH]_(n)CH₂R)), 174 (s,bipyNiN(H)C(H)R¹³ C(O)N[CH(R)¹³C(O)NH]_(n)CH₂R).

Polymerization of Glu-NCA with (2,2′-bipyridyl)Ni(COD)

In the dry box, Glu NCA (50 mg, 0.2 mmol) was dissolved in DMF (0.5 mL)and placed in a 25 mL reaction tube which could be sealed with a Teflonstopcock. An aliquot of bipyNi(COD) (50 ml of a 40 mM solution in DMF)was then added via syringe to the flask. A stirbar was added and theflask was sealed, removed from the dry box, and placed in a thermostated25° C. bath for 16 hours. Polymer was isolated by addition of thereaction mixture to methanol containing HCl (1 mM) causing precipitationof the polymer. The polymer was then dissolved in THF and reprecipitatedby addition to methanol. The polymer was dried in vacuo to give a whitestringy solid, PBLG (41 mg, 98% yield). ¹³C {¹H} NMR, ¹H NMR, and FTIRspectra of this material were identical to data found for authenticsamples of PBLG. GPC of the polymer in 0.1M LiBr in DMF at 60° C.:M_(n)=22,000; M_(w)/M_(n)=1.05.

As an illustrative embodiment of the invention, diblock copolymerscomposed of amino acid components g-benzyl-L-glutamate ande-carbobenzyloxy-L-lysine were synthesized. The polymers were preparedby addition of Lys-NCA to bipyNi(COD) in DMF to afford livingpoly(e-carbobenzyloxy-L-lysine), PZLL, chains with organometallicend-groups capable of further chain growth. Glu-NCA was added to thesepolymers to yield the PBLG-PZLL block copolypeptides. The evolution ofmolecular weight through each stage of monomer addition was analyzedusing gel permeation chromatography (GPC) and data are given in Table 1below. Molecular weight was found to increase as expected upon growth ofeach block of copolymer while polydispersity remained low, indicative ofsuccessful copolymer formation. A. Noshay, et al., Block Copolymers,Academic Press, New York, (1977).

The chromatograms of the block copolypeptides showed single sharp peaksillustrating the narrow distribution of chain lengths (See FIG. 2).Copolypeptide compositions were easily adjusted by variation of monomerfeed compositions, both being equivalent. Successful preparation ofcopolypeptides of reverse sequence (i.e. PZLL-PBLG) and of triblockstructure (e.g. PBLG_(0.39)-b-PZLL_(0.22)-b-PBLG_(0.39); M_(n)=256,000,M_(w)/M_(n)=1.15) illustrate the potential for sequence control usingthe nickel initiator.

Block copolymerizations were not restricted to the highly solublepolypeptides PBLG and PZLL. Copolypeptides containing L-leucine andL-proline, both of which form homopolymers which are insoluble in mostorganic solvents (e.g. DMF) were prepared. Data for thesecopolymerizations are given in Table 4 below. Because of thesolubilizing effect of the PBLG and PZLL blocks, all of the productswere soluble in the reaction media indicating the absence of anyhomopolymer contaminants. The block copolymers containing L-leucine werefound to be strongly associating in 0.1M LiBr in DMF, a good solvent forPBLG and PZLL. Once deprotected, the assembly properties of thesematerials are expected to make them useful as tissue engineeringscaffolds, drug carriers, and morphology-directing components inbiomimetic composite formation.

TABLE 4 Preparation and analysis of block copolypeptides. Polymerizationinitiator was bipyNi(COD) in DMF in all cases. Molecular weight (Mn) andpolydispersity (M_(w)/M_(n)) were determined by tandem GPC/lightscattering in 0.1M LiBr in DMF at 60° C. using dn/dc values measured inthis solvent at I0 = 633 nm. First Diblock segment† Copolymer‡ FirstSecond M_(w)/ M_(w)/ Yield Monomer* Monomer* M_(n) M_(n) M_(n) M_(n)(%)§ 52 Lys-NCA 181 Glu-  15,000∥ 1.12 66,000¶ 1.21 95 NCA 90 Glu-NCA 78Lys-NCA 28,500# 1.12  52,700** 1.13 93 104 Lys-NCA 40 Leu-NCA  29500∥1.13  34,000∥ 1.20 93 182 Glu-NCA 90 Pro-NCA 57,600# 1.07 86,000# 1.1492 120 Glu-NCA 40 Leu-NCA 38,000# 1.08 79,000# 1.13 96 *First and secondmonomers added stepwise to the initiator; number indicates equivalentsof monomer per bipyNi(COD). Leu-NCA = L-leucine-N-carboxyanhydride.Pro-NCA = L-proline-N-carboxyanhydride. †Molecular weight andpolydispersity after polymerization of the first monomer. ‡Molecularweight and polydispersity of the complete block copolymer. §Totalisolated yield of block copolymer. ∥dn/dc = 0.123 mL/g. ¶dn/dc = 0.108mL/g. #dn/dc = 0.104 mL/g. **dn/dc = 0.115 mL/g.

The initiators described above were generated usingbis-1,5-cyclooctadiene nickel (Ni(COD)₂) as the nickel source and2,2′-bipyridyl (bipy) as the donor ligand component in tetrahydrofuran(THF) solvent. Other sources of zerovalent nickel (e.g. Ni(CO)₄) as wellas other donor ligands (e.g. PR₃ [R=Me, Et, Bu, cyclohexyl, phenyl],R₂PCH₂CH₂PR₂ [R=Me, phenyl], a,a′-diimine ligands [1,10-phenanthroline,neocuproine], diamine ligands [tetramethylethylene diamine], andisocyanide ligands [tert-butyl isocyanide]) can be used to initiatethese polymerizations. The use of other sources of zerovalent nickel(e.g. nickel-olefin complexes, nickel-carbonyl complexes,nickel-isocyanide or cyanide complexes, and nickel nitrogen orphosphorous donor ligand complexes) are possible embodiments whichshould not be interpreted as going beyond the concept of this invention.Likewise, the use of other donor ligands (nitrogen or phosphorous basedin particular) or polymerization solvents are logical extensions of thiswork. Finally, other transition metals, specifically palladium,platinum, cobalt, rhodium, iridium and iron are also able to polymerizeNCA monomers. The use of metals in “Group VIII” (i.e. Co, Rh, Ir, Ni,Pd, Pt, Fe, Ru, Os) are thus additional potential embodiments of thisinvention.

Illustrative Diblock and Triblock Copolypeptides and their Synthesis 1.Poly(e-benzyloxycarbonyl-L-Lysine-block-g-benzyl-L-glutamate),PZLL-b-PBLG, Diblock Copolymer

In the dry box, Glu NCA (50 mg, 0.19 mmol) was dissolved indimethylformamide (DMF) (0.5 mL) and placed in a 25 mL reaction tubewhich could be sealed with a Teflon stopcock. An aliquot of(2,2′-bipyridyl)Ni(COD) (50 ml of a 40 mM solution in DMF, prepared bymixing equimolar amounts of 2,2′-bipyridyl and Ni(COD)₂) was then addedvia syringe to the flask. A stirbar was added, the flask was sealed andthen stirred for 16 hours. An aliquot (50 mL) was removed from thepolymerization for GPC analysis (M_(n)=28,500; M_(w)/M_(n)=1.12).e-benzyloxycarbonyl-L-Lysine-N-carboxyanhydride, Lys-NCA, (50 mg, 0.16mmol) dissolved in dimethylformamide (DMF) (0.5 mL) was then added tothe reaction mixture. After stirring for an additional 16 h, polymer wasisolated by addition of the reaction mixture to methanol containing HCl(1 mM) causing precipitation of the polymer. The polymer was thendissolved in THF and reprecipitated by addition to methanol. The polymerwas dried in vacuo to give a white solid, PZLL-b-PBLG (79 mg, 93%yield). ¹³C {¹H} NMR, ¹H NMR, and FTIR spectra of this material wereidentical to a combination of data found for authentic individualsamples of PBLG and PZLL. GPC of the block copolymer in 0.1M LiBr in DMFat 60° C.: M_(n)=52,700; M_(w)/M_(n)=1.13.

2. Poly (M-benzyloxycarbonyl-L-Lysine-block-K-benzyl-L-glutamate),PZLL-b-PBLG, Diblock Copolymer using (PMe₃)₄Co

In the dry box, Glu NCA (50 mg, 0.19 mmol) was dissolved in DMF (0.5 mL)and placed in a 15 mL reaction tube which could be sealed with a TEFLON™stopper. An aliquot of (PMe₃)₄)Co (50 TL of a 40 mM solution in DMF:THF(1:1)) was then added via syringe to the flask. A stirbar was added, theflask was scaled and then stirred for 16 h. An aliquot (50 TL) wasremoved from the polymerization for GPC analysis (M_(n)=21,500;M_(w)/M_(n)=1.12). Lys-NCA, (50 mg, 0.16 mmol) dissolved in DMF (0.5 mL)was then added to the reaction mixture. After stirring for an additional16 h, polymer was isolated by addition of the reaction mixture tomethanol containing HCl (1 mM) causing precipitation of the polymer. Thepolymer was then dissolved in THF and reprecipitated by addition tomethanol. The polymer was dried in vacuo to give a white solid,PZLL-b-PBLG (82 mg, 97% yield). ¹³C {¹H} NMR, ¹H NMR, and FTIR spectraof this material were identical to a combination of data found forauthentic individual samples of PBLG and PZLL. GPC of the blockcopolymer in 0.1M LiBr in DMF at 60° C.; M_(n)-44,700; M_(w)/M_(n)=1.13.

3.Poly(q-benzyl-L-glutamate-block-e-benzyloxycarbonyl-L-Lysine-b/ock-q-benzyl-L-glutamate)Triblock Copolymer

In the dry box, Glu NCA (250 mg, 0.95 mmol) was dissolved indimethylformamide (DMF) (1.5 mL) and placed in a 25 mL reaction tubewhich could be sealed with a Teflon stopcock. An aliquot of(2,2′-bipyridyl)Ni(COD) (50 ml of a 40 mM solution in DMF, prepared bymixing equimolar amounts of 2,2′-bipyridyl and Ni(COD)₂) was then addedvia syringe to the flask. A stirbar was added, the flask was sealed andthen stirred for 16 hours. An aliquot (50 mL) was removed from thepolymerization for GPC analysis (M_(n)=100, 100; M_(w)/M_(n)=1.11).Lys-NCA, (125 mg, 0.42 mmol) dissolved in dimethylformamide (DMF) (0.5mL) was then added to the reaction mixture, which was stirred for 16 h.A second aliquot (50 mL) was removed from the polymerization for GPCanalysis (M_(n)=156, 200; M_(w)/M_(n)=1.12). Finally, Glu-NCA, (250 mg,0.95 mmol) dissolved in dimethylformamide (DMF) (1.5 mL) was then addedto the reaction mixture. After stirring for an additional 16 h, polymerwas isolated by addition of the reaction mixture to methanol containingHCl (1 mM) causing precipitation of the polymer. The polymer was thendissolved in THF and reprecipitated by addition to methanol. The polymerwas dried in vacuo to give a white solid, PBLG-b-PZLL-b-PBLG (505 mg,96% yield). ¹³C {¹H} NMR, ¹H NMR, and FTIR spectra of this material wereidentical to a combination of data found for authentic individualsamples of PBLG and PZLL. GPC of the block copolymer in 0.1M LiBr in DMFat 60° C.: M_(n)=256, 300; M_(w)/M_(n)=1.15.

4. General Preparation of Block Copolypeptides with Metal Initiators

Other diblock and triblock copolymers were prepared by a procedureidentical to that described above for either PZLL-b-PBLG andPBLG-b-PZLL-b-PBLG, except that either different monomers, or differentamounts of monomers, were used for the individual polymerizationreactions. Examples are given in Tables 4 (above) and Table 5 (below).The nature of the amino acid monomer was found to be unimportant inlimiting the effectiveness of these polymerizations. All amino acid NCAstried were incorporated into block copolypeptides in any sequentialorder, as determined by the order of addition to the initiator.Representative monomers include, but are not limited to: the naturallyoccurring L-amino acids, naturally occurring D-amino acids,a-disubstituted a-amino acids, racemic a-amino acids, and synthetica-amino acids. Block copolypeptides could be prepared using initiatorsother than (2,2′-bipyridyl)Ni(COD). The initiators given in Tables 7 and8 below (except those that gave no yield of polymer) all were able toprepare block copolypeptides.

TABLE 5 Preparation and analysis of triblock copolypeptides.Polymerization initiator was bipyNi(COD) in DMF in all cases. Molecularweight (Mn) and polydispersity (M_(w)/M_(n)) were determined by tandemGPC/light scattering in 0.1M LiBr in DMF at 60° C. using dn/dc valuesmeasured in this solvent at I0 = 633 nm. Monomers and order of addition*Diblock Triblock 1^(st) 2^(nd) 3^(rd) 1^(st) segment^(†) segment^(‡)copolymer^(∥) monomer monomer monomer M_(n) M_(w)/M_(n) M_(n)M_(w)/M_(n) M_(n) M_(w)/M_(n) Yield^(§) 450 Glu 200 Lys 450 Glu 100 1.11156 1.12 256 1.15 96 130 Glu 120 Ala 130 Glu 34 1.07 47 1.14 82 1.20 94250 Glu 130 Leu 250 Glu 68 1.12 83 1.18 152 1.20 97 250 Glu 300 Pro 250Glu 67 1.10 98 1.17 167 1.18 96 *First, second and third monomers addedstepwise to the initiator; number indicates equivalents of monomer perbipyNi(COD). Lys = Lys-NCA. Glu = Glu-NCA. Ala =L-alanine-N-carboxyanhydride. Leu = L-leucine-N-carboxyanhydride. Pro =L-proline-N-carboxyanhydride. ^(†)Molecular weight (×10⁻³) andpolydispersity after polymerization of the first monomer. ^(‡)Molecularweight (×10⁻³) and polydispersity after polymerization of the secondmonomer. ^(∥)Molecular weight (×10⁻³) and polydispersity of the completetriblock copolymer. ^(§)Total isolated yield (%) of triblock copolymer.

Example 4 General Initiator Features; Assessment of Multiple Initiatorsand Effect of Chemical Structure of Efficiency; Effects of ReactionConditions on Polymerization: and Initiator Mediated Block CopolypeptideSynthesis

General Protocols and Reagents.

Infrared spectra were recorded on a Perkin Elmer 1605 FTIRSpectrophotometer calibrated using polystyrene film. Optical rotationswere measured on a Perkin Elmer Model 141 Polarimeter using a 1 mLvolume cell (1 dm length). NMR spectra and bulk magnetic susceptibilitymeasurements (Evans method) were measured on a Bruker AMX 500 MHzspectrometer. D. F. Evans, J. Chem. Soc., 2003-2009 (1959); J. K.Becconsal, J. Mol. Phys., 15:129-135 (1968). C, H, N elemental analyseswere performed by the Microanalytical Laboratory of the University ofCalifornia, Berkeley Chemistry Department. Metal analyses were conductedusing a Thermo Jarrell Ash IRIS HR ICP analyzer. Chemicals were obtainedfrom commercial suppliers and used without purification unless otherwisestated. (COD)₂Ni was obtained from Strem Chemical Co., and¹³C₁-L-leucine and ¹³C-phosgene were obtained from Cambridge IsotopeLabs. L-leucine isoamylamide hydrochloride, g-benzyl-L-glutamate NCA andL-leucine NCA were prepared according to literature procedures. M.Bodanszky, et al., The practice of Peptide Synthesis, 2^(nd) Ed.,Springer, Berlin/Heidelberg, (1994); E. R. Blout, et al., J. Am. Chem.Soc., 78:941-950 (1956); H. Kanazawa, et al., Bull. Chem. Soc. Jpn.,51:2205-2208 (1978). Hexanes, THF, and THF-d₈ were purified bydistillation from sodium benzophenone ketyl. DMF and DMF-d₇ werepurified by drying over 4 Å molecular sieves followed by vacuumdistillation.

General Features for Formation of Active Metal Initiators

The efficient, controlled polymerization of NCAs using transition metalcompounds requires the general formation of an amido-containing 5- or6-membered metallacycle (FIG. 6), which is the active intermediate inthe polymerizations. With regard to results described in thisdisclosure, these metallacycles are formed by reaction of 1 or 2equivalents of an NCA with a metal complex, which is capable ofundergoing an oxidative-addition reaction where its valence formallyincreases by two. A variety of NCAs can be used for this reaction (i.e.L-leucine NCA, Glu-NCA, and L-phenylalanine NCA) and there is no reasonwhy any NCA of general structure shown in FIG. 6 would not work for thisreaction. The metals which most commonly undergo two-electronoxidative-addition reactions are those in Group VIII (i.e. Fe, Ru, Os,Co, Rh, Ir, Ni, Pd, Pt) and hence these are the metals studied mostextensively. Collman, J. P.; Roper, W. R. Adv. Orgmet, Chem., 1968, 7,53-94. Amido-containing metallacycles can be formed with Fe, Co, Rh, Irand Ni, and that these complexes give controlled polymerization of NCAs.Pd and Pt complexes are also able to promote polymerization of NCAs.Virtually any low-valent transition metal (i.e. a metal in a lowoxidation state) with the proper combination of electron donor ligand(s)can react with NCAs to yield amido-containing metallacyclicintermediates which could act as active polymerization initiators. Othermetals which clearly fall into this category are Au, Mn, Cr, Mo, W, andV.

The range of substituents (R) which can be placed on theamido-containing metallacycles was investigated. These include the sidechain functions found in amino acids themselves (e.g. R═CH₂C₆H₅ fromphenylalanine, R═CH₂CH(CH₃)₂, or R═CH₂CH₂CO₂CH₂C₆H₅ fromg-benzylglutamate), and should thus include any organic moiety attachedto an a-amino acid.

Determination of Initiator Efficiency.

Efficiencies were quantified by measurement of product polymer molecularweights and molecular weight distributions, and measurement ofpolymerization reaction rates. Polymer molecular weights and molecularweight distributions were measured using tandem gel permeationchromatography/light scattering (GPC/LS) which was performed on aSpectra Physics Isochrom liquid chromatograph pump equipped with a WyattDAWN DSP light scattering detector and Wyatt Optilab DSPinteroferometric refractometer. Separations were effected by 10⁵ Å and10³ Å Phenomenex 5μ columns using 0.1M LiBr in DMF eluent at 60° C.Polymerization reaction rates were obtained from kinetic data which weremeasured by periodically removing aliquots from a thermostatedpolymerization of Glu-NCA, diluting these (10-fold) with anhydrouschloroform to a known volume, and recording the intensity of theunreacted anhydride stretch at 1790 cm⁻¹ in the solution by FTIRspectroscopy. NCA concentrations were determined by use of an empiricalcalibration curve (transmittance vs. concentration) of Glu-NCA inchloroform. Plots of log (concentration) versus time gave pseudo firstorder polymerization rates for the different initiators.

(S)-[NiNHC(H)RC(O)NCH₂R]_(x), R=—CH₂CH₂C(O)OCH₂C₆H₅: NiGlu₂

In the dry box, Glu NCA (15 mg, 0.058 mmol) was dissolved in THF (0.5mL) and added to a stirred homogeneous mixture of PPh₃ (31 mg, 0.12mmol) and (COD)₂Ni (16 mg, 0.058 mmol) in THF (1.5 mL). The red/brownsolution was stirred for 24 hours, after which the solvent was removedin vacuo to leave a dark red oily solid. This was extracted with hexanes(3×5 mL) to yield a red/brown hexanes solution and a yellow solid.Evaporation of the hexanes solution gave a red oil containing(PPh₃)₂Ni(CO)₂ [IR (THF): 2000, 1939 cm⁻¹ (nCO, vs); 18 mg. J. Chatt, etal., J. Chem. Soc., 1378-1389 (1960). IR (CH₂ClCH₂Cl): 1994, 1933cm⁻¹)], and drying of the solid gave the product as a yellow powder (10mg, 75% yield). An ¹H NMR spectrum could not be obtained in THF-d₈, mostlikely because of paramagnetism of the complex (only broad lines for thebenzyl ester groups were observed). m_(eff) (THF, 293 K)=1.08 m_(B).Osmotic molecular weight in THF (vs. ferrocene; ca. 7 mg/mL): 910 g/mol;this corresponds to a degree of aggregation of 1.94. IR (THF): 3281 cm⁻¹(nNH, s br), 1734 cm⁻¹ (nCO, ester, vs), 1577 cm⁻¹ (nCO, amidate, vs).Anal. calcd. for NiC₂₃H₂₆N₂O₅: 58.87% C, 5.59% H, 5.96% N. found: 59.07%C, 5.67% H, 5.56% N. [a]_(D) ²⁰ (THF, c=−0.0034)=−71.

(S)-[NiNHC(H)RC(O)NCH₂R]_(x), R=—CH₂C₆H₅ NiPhe₂

In the dry box, L-phenylalanine NCA (45 mg, 0.24 mmol) was dissolved inTHF (0.5 mL) and added to a stirred homogeneous mixture of PPh₃ (124 mg,0.48 mmol) and (COD)₂Ni (64 mg, 0.24 mmol) in THF (1.5 mL). Thered/brown solution was stirred for 24 hours, after which the solvent wasremoved in vacuo to leave a dark red oily solid. This was extracted withcold hexanes (0° C., 3×2 mL) to yield a red/brown hexanes solution and apale orange solid. Evaporation of the hexanes solution gave a red oilcontaining (PPh₃)₂Ni(CO)₂ [IR (THF): 2000, 1939 cm⁻¹ (nCO, vs)], anddrying of the solid gave an orange powder which could be purified byprecipitation from THF/hexanes to give (S)-[NiNHC(H)RC(O)NCH₂R]_(x),R=—CH₂C₆H₅ as a yellow powder (31 mg, 80% yield). An ¹H NMR spectrumcould not be obtained in THF-d₈, most likely because of paramagnetism ofthe complex. IR (THF): 3290 cm⁻¹ (nNH, s br), 1574 cm⁻¹ (nCO, amidate,vs). [a]_(D) ²⁰ (THF, c=0.001)=−170.

(S)-(2,2′-bipyridyl)NiNHC(H)RC(O)NCH₂R, R=—CH₂CH₂C(O)OCH₂C₆H₅;(2,2′-bipyridyl)NiGlu₂

In the dry box, a yellow solution of NiGlu₂ (40 mg, 0.085 mmol) in DMF(0.5 mL) was added to a solution of 2,2′-bipyridyl (54 mg, 0.35 mmol) inDMF (0.5 mL). The homogeneous mixture was stirred for 2 d at 50° C.,during which the color changed from yellow to blood red. THF (1 mL) andtoluene (5 mL) were layered onto this solution resulting inprecipitation of a red powder. This powder was reprecipitated fromDMF/THF/toluene (1:2:10) two additional times to give(2,2′-bipyridyl)NiGlu₂ as a red powder (49 mg, 92% yield). An ¹H NMRspectrum could not be obtained in THF-d₈, most likely because ofparamagnetism of the complex (only broad lines for the benzyl estergroups were observed). IR (THF): 3281 cm⁻¹ (nNH, s br), 1732 cm⁻¹ (nCO,ester, vs), 1597 cm⁻¹ (nCO, amidate, vs). Anal. calcd. for NiC₃₃H₃₄N₄O₅:63.37% C, 5.49% H, 8.95% N; found: 63.72% C, 5.49% H, 8.86% N. [a]_(D)²⁰ (THF, c=0.001)=−135.

Preparation of Other L₂NiGlu₂ and L₂NiPhe₂ Initiators

The procedures for synthesis of these compounds were identical to thatdescribed for preparation of (2,2′-bipyridyl)NiGlu₂ except forsubstitution of different ligands (L₂) for 2,2′-bipyridyl or the use ofNiPhe₂ instead of NiGlu₂. The range of ligands included phen, LiCN, andtmeda. All of the complexes gave satisfactory analysis.

1. (PMe₃)₂FePhe₂

In the dry box, L-phenylalanine NCA (32 mg, 0.16 mmol) was dissolved inTHF (0.5 mL) and added to a stirred homogeneous solution of (PMe₃)₄Fe(30 mg, 0.083 mmol) in Et₂O (4 mL). The pale orange solution was stirredfor 24 hours, after which the resulting off-white precipitate wasisolated by centrifugation. This solid was washed with Et₂O (3×5 mL) andthen dried to give an off-white powder. The powder was purified bydissolving in THF and precipitating with hexanes (36 mg, 91%). An ¹H NMRspectrum could not be obtained in THF-d₈, most likely because ofparamagnetism of the complex (only broad lines for the phenyl groupswere observed). IR (THF): 3296 cm⁻¹ (nNH, s br), 1603 cm⁻¹ (nCO,amidate, vs).

2. (tBuNC)₂FePhe₂

In the dry box, (PMe₃)₂FePhe₂ (20 mg, 0.042 mmol) was dissolved in THF(2 mL) and mixed with tBuNC (24 mL, 0.252 mmol) in THF (2 mL). Thesolution was stirred overnight during which it slowly turned from brownto yellow. The product was isolated by repeated precipitation of ayellow powder from THF by addition to hexanes. Drying gave a yellowsolid (19 mg, 94%). IR (THF): 3289 cm⁻¹ (nNH, s br), 2150 cm⁻¹ (nNC,tBuNC, vs), 1626 cm⁻¹ (nCO, amidate, vs).

3. (2,2′-bipyridyl)FePhe₂

In the dry box, (PMe₃)₂FePhe₂ (20 mg, 0.042 mmol) was dissolved in THF(2 mL) and mixed with tBuNC (33 mg, 0.168 mmol) in THF (2 mL). Thesolution was stirred overnight during which it slowly turned from brownto deep red. The product was isolated by repeated precipitation of a redpowder from DMF:THF (1:1) by addition to hexanes. Drying gave a redsolid (18 mg, 89%). IR (THF): 3291 cm⁻¹ (nNH, s br), 1600 cm⁻¹ (nCO,amidate, vs).

Polymerization of Glu-NCA with (2,2′-bipyridyl)Ni(COD)

In the dry box, Glu NCA (50 mg, 0.2 mmol) was dissolved intetrahydrofuran (THF) (0.5 mL) and placed in a 25 mL reaction tube whichcould be sealed with a Teflon stopcock. An aliquot of(2,2′-bipyridyl)Ni(COD) (50 ml of a 40 mM solution in THF, prepared bymixing equimolar amounts of 2,2′-bipyridyl and Ni(COD)₂) was then addedvia syringe to the flask. A stirbar was added and the flask was sealed,removed from the dry box, and stirred in a thermostated 25° C. bath for16 hours. Polymer was isolated by addition of the reaction mixture tomethanol containing HCl (1 mM) causing precipitation of the polymer. Thepolymer was then dissolved in THF and reprecipitated by addition tomethanol. The polymer was dried in vacuo to give a white solid, PBLG (41mg, 98% yield). ¹³C {¹H} NMR, ¹H NMR, and FTIR spectra of this materialwere identical to data found for authentic samples of PBLG. H. Block,Poly(g-benzyl-L-glutamate) and Other Glutamic Acid Containing Polymers,Gordon and Breach, New York, (1983). GPC of the polymer in 0.1M LiBr inDMF at 60° C.: M_(n)=98,100; M_(w)/M_(n)=1.15.

General Polymerization of Glu-NCA with (2,2′-bipyridyl)Ni(COD) inDifferent Solvents

The procedure followed was identical to that used for the(2,2′-bipyridyl)Ni(COD) in THF except for substitution of differentsolvents for THF. The range of other solvents included: toluene,dioxane, acetonitrile, ethyl acetate, and DMF. The results of thesepolymerizations are given in Table 6 below. The initiator efficiencieswere determined by analysis of polymer yields, proximity of the foundmolecular weights to the theoretical values, and the narrowness of themolecular weight distributions.

TABLE 6 Effect of solvent on polymerizations of Glu-NCA using2,2′-bipyridylNi(COD) initiator. Moles monomer:moles initiator = 180:1.All polymerizations were run at 20° C., for 16 hours under nitrogenatmosphere. Notebook # Solvent Yield (%) M_(n) M_(w)/M_(n) 3-23 Ethylacetate 99 109,000 1.12 2-148 Toluene 97 146,000 1.11 3-23 Dioxane 96126,000 1.20 3-23 Acetonitrile 62 75,000 1.45 2-144 THF 96 142,000 1.052-151 DMF 97 40,000 1.19

General Polymerization of Glu-NCA with (L₂)Ni(COD) Initiators

The procedure followed was identical to that used for the(2,2′-bipyridyl)Ni(COD) initiator except for substitution of differentligand molecules (L₂) for 2,2′-bipyridyl. The range of ligands (L₂)included: tricyclohexylphosphine (PCy₃, 1 and 2 equivalents per metal),tert-butyl isocyanide (tBuNC, 2 and 4 equivalents), lithium cyanide (2equivalents), trimethylphosphine (PMe₃, 2 equivalents),triethylphosphine (PEt₃, 2 equivalents), tributylphosphine (PBu₃, 2equivalents), triphenylphosphine (PPh₃, 1 and 2 equivalents),1,2-bis(diphenylphosphino)ethane (DIPHOS),1,2-bis(dimethylphosphino)ethane (dmpe), tetramethylethylenediamine(tmeda), (−)-sparteine, 1,10-phenanthroline (phen), neocuproine (ncp),as well as the compounds shown in FIG. 5. The results of thesepolymerizations are given in Table 7 below. The initiator efficiencieswere determined by analysis of polymer yields, proximity of the foundmolecular weights to the theoretical values, and the narrowness of themolecular weight distributions.

TABLE 7 Effect of Ligands on L₂Ni(COD) initiators for polymerizations ofGlu-NCA. [M]:[I] = moles monomer:moles initiator. All polymerizationswere run at 20° C. for 16 hours under nitrogen atmosphere. L₂:Ni(COD)₂ =1:1 unless specified otherwise. Note- Yield M_(n) × book # Ligand (L₂)[M]:[I] Solvent (%) 10⁻³ M_(w)/M_(n) 2-144 2,2 = -bipyridyl 180 THF 96142 1.05 2-148 DIPRIM ″ ″ 60 165 1.21 2-148 dmpe ″ ″ 90 275 1.04 2-148COD ″ ″ 0 — — 2-151 DMIM ″ ″ 0 — — 2-151 2 PPh₂ ″ ″ 0 — — 2-151 1 PPh₂ ″″ 78 126 1.26 2-151 phen  90 ″ 94 151 1.15 2-151 ncp ″ ″ 94 293 1.17 3-2DPIM ″ ″ 0 — — 3-2 DPOX ″ ″ 96 189 1.06 3-2 2 PMe₃ ″ ″ 94 244 1.16 3-10tmeda ″ ″ 96 305 1.09 3-11 DIPHOS ″ ″ 0 — — 3-21 2- PCy₃ ″ ″ 0 — — 3-211 PCy₃ ″ ″ 0 — — 3-34 2 t-BuNC ″ ″ 76 218 1.09 3-34 4 t-BuNC ″ ″ 74 1901.15 3-37 2 PEt₃ ″ ″ 92 251 1.08 3-37 2 PBu₃ ″ ″ 88 196 1.14 JJ-Rep(-)sparteine 200 ″ 92 174 1.05 JJ-Rep TMOX ″ ″ 98 170 1.14 JJ-RepDMOX-py ″ ″ 90 157 1.03 2-151 2,2 = -bipyridyl 152 DMF 97  35 1.14 3-11tmeda  90 ″ 96  87 1.36 3-11 dmpe ″ ″ 96  60 1.33 3-11 phen ″ ″ 98  411.21 3-11 ncp ″ ″ 99  48 1.45 3-11 DIPHOS ″ ″ 94 100 1.50 3-21 1 PCy₃ ″″ 35  46 1.18

General Polymerization of Glu-NCA with Other Transition Metal Initiators

The procedure followed was identical to that used for the(2,2′-bipyridyl)Ni(COD) initiator except for substitution of differentmetal complexes for (2,2′-bipyridyl)Ni(COD). The range of metalcomplexes included: (2,2′-bipyridyl)Ni(CO)₂;(S)-[NiNHC(H)RC(O)NCH₂R]_(x), R=—CH₂CH₂C(O)OCH₂C₆H₅ (NiGlu₂);(S)-(2,2′-bipyridyl)NiNHC(H)RC(O)NCH₂R, R=—CH₂CH₂C(O)OCH₂C₆H₅(2,2′-bipyridylNiGlu₂); (S)-Li₂(CN)₂NiNHC(H)RC(O)NCH₂R,R=—CH₂CH₂C(O)OCH₂C₆H₅ (Li₂(CN)₂NiGlu₂); (S)-(phen)NiNHC(H)RC(O)NCH₂R,R=—CH₂CH₂C(O)OCH₂C₆H₅ (phenNiGlu₂); (S)-(phen)NiNHC(H)RC(O)NCH₂R,R=—CH₂C₆H₅ (phenNiPhe₂); (S)-(tmeda)NiNHC(H)RC(O)NCH₂R, R=—CH₂C₆H₅(tmedaNiPhe₂); dmpeCoPhe₂; (PMe₃)₂CoPhe₂; (PMe₃)₄Co; dmpeRhCl; dmpeIrCl;h⁵-C₅H₅CO(CO)₂ (CpCo(CO)₂); (2,2′-bipyridylCo(CO)₂)₂; ((PPh₃)₂Co(CO)₂)₂;(PMe₃)₄Fe; (2,2′-bipyridyl)₂Fe; (S)-(PMe₃)₂FeNHC(H)RC(O)NCH₂R,R=—CH₂C₆H₅ ((PMe₃)₂FePhe₂); (S)-(tBuNC)₂FeNHC(H)RC(O)NCH₂R, R=—CH₂C₆H₅((tBuNC)₂FePhe₂); (S)-(2,2′-bipyridyl)FeNHC(H)RC(O)NCH₂R, R=—CH₂C₆H₅((2,2′-bipyridyl)FePhe₂); (PPh₃)₄Pd;tris(dibenzylideneacetone)dipalladium (Pd₂(DBA)₃) plus 4 equivalents ofPEt₃; (PEt₃)₂Pt(COD); and (dmpe)₂Co. The results of thesepolymerizations are given in Table 8 below. The initiator efficiencieswere determined by analysis of polymer yields, proximity of the foundmolecular weights to the theoretical values, and the narrowness of themolecular weight distributions.

Polymerization of Glu-NCA with (PMe₃)₄Co

In the dry box, Glu NCA (50 mg, 0.2 mmol) was dissolved in DMF (0.5 mL)and placed in a 15 mL reaction tube which could be sealed with a TEFLON™stopper. An aliquot of (PMe₃)₄)Co (50 TL of a 40 mM solution in DMF:THF(1:1)) was then added via syringe to the flask. A stirbar was added andthe flask was sealed, removed from the dry box, and stirred in athermostated 25° C. bath for 16 h. Polymer was isolated by addition ofthe reaction mixture to methanol containing HCl (1 mM) causingprecipitation of the polymer. The polymer was then dissolved in THF andreprecipitated by addition to methanol. The polymer was dried in vacuoto give a white solid, PBLG (42 mg, 99% yield). ¹³C {¹H} NMR, ¹H NMR,and FTIR spectra of this material were identical to data found forauthentic samples of PBLG. GPC of the polymer in 0.1M LiBr in DMF at 60°C.: M_(n)=21,600; M_(w)/M_(n)=1.11.

(S)-[CoNHC(H)RC(0)NCH₂R]₂₅ R═CH₂C₆H₆, CoPhe₂

In the dry box, Phe NCA (9.0 mg, 0.046 mmol) was dissolved in THF (0.5mL) and added to a stirred homogeneous solution of (PPh₃)₃Co(N₂) (40 mg,0.046 mmol) in THF (1.5 mL). The red/brown solution was stirred for 24h, after which the solvent was removed in vacuo to leave a red/orangeoily solid. This was extracted with hexanes (3×5 mL) to yield an orangehexanes solution and a tan solid. Evaporation of the hexanes solutiongave a brown oil containing [(PPh₃)₃Co(CO)]₂ [IR (THF): 1909, 1875 cm⁻¹(nCO, vs); 15 mg; Literature: IR (KBr): 1904, 1877 cm⁻¹)], and drying ofthe solid gave the product as a tan powder (11 mg, 74% yield). An ¹H NMRspectrum could not be obtained in THF-d₈ most likely because ofparamagnetism of the complex (only broad lines for the phenyl rings wereobserved). IR (THF): 3310 cm⁻¹ (nNH, s br), 1600 cm⁻¹ (nCO, amidate,vs).

(S)-(dmpe)CoNHC(H)RC(O)NCH₂R, R=—CH₂C₆H₆ dmpeCoPhe₂

In the dry box, a light brown solution of 1 (40 mg, 0.12 mmol) in DMF(0.5 mL) was added to a solution of bis(dimethylphosphino)ethane, dmpe,(35 TL, 0.21 mmol) in DMF (0.5 mL). The homogeneous mixture was stirredfor 2 d at 50° C., during which the color changed from yellow toorange/red. THF (1 mL) and toluene (5 mL) were layered onto thissolution resulting in separation of a brown oil. This oil was isolatedfrom DMF/THF/toluene (1:2:10) two additional times to give the product(49 mg, 86% yield). An ¹H NMR spectrum could not be obtained in THF-d₈,most likely because of paramagnetism of the complex (only broad linesfor the phenyl and methyl groups were observed). IR (THF): 3295 cm⁻¹(nN11, s br), 1603 cm⁻¹ (nCO, amidate, vs).

Polymerization of Glu-NCA using dmpeCoPhe₂

In the dry box, Glu NCA (50 mg, 0.2 mmol) was dissolved in DMF (0.5 mL)and placed in a 15 mL reaction tube which could be sealed with a TEFLON™stopper. An aliquot of dmpeCoPhe₂ (50 TL of a 40 mM solution in DMF) wasthen added via syringe to the flask. A stirbar was added and the flaskwas sealed, removed from the dry box, and stirred in a thermostated 25°C. bath for 16 h. Polymer was isolated by addition of the reactionmixture to methanol containing HCl (1 mM) causing precipitation of thepolymer. The polymer was then dissolved in THF and reprecipitated byaddition to methanol. The polymer was dried in vacuo to give a whitesolid, PBLG (41 mg, 98% yield). ¹³C {¹H} NMR, ¹H NMR, and FTIR spectraof this material were identical to data found for authentic samples ofPBLG. GPC of the polymer in 0.1M LiBr in DMF at 60° C.: M_(n)=20,900:M_(w)/M_(n)=1.07.

TABLE 8 Efficiency of different transition metal initiators forpolymerization of Glu-NCA. [M]:[I] = moles monomer:moles initiator. Allpolymerizations were run at 20° C. for 16 hours under nitrogenatmosphere. Yield Notebook # Metal complex [M]:[I] Solvent (%) M_(n) ×10⁻³ M_(w)/M_(n) 3-40 2,2′-bipyridylNi(CO)₂ 50 THF 54 38 1.38 3-45NiGlu₂ ″ DMF 84 35 1.26 3-34 2,2′-bipyridylNiGlu₂ ″ THF 97 142 1.12 3-58Li₂(CN)₂NiGlu₂ ″ ″ 96 100 1.27 3-64 phenNiGlu₂ 30 ″ 97 83 1.15 ″phenNiPhe₂ ″ ″ 98 98 1.11 3-62 tmedaNiPhe₂ ″ ″ 96 100 1.10 3-612,2′-bipyridylNiGlu₂ 90 DMF 94 60 1.18 3-64 phenNiGlu₂ ″ ″ 97 36 1.153-68 phenNiPhe₂ ″ CH₂Cl₂ 93 86 1.09 3-58 CpCo(CO ″ THF 0 — — 3-362,2′-bipyridylCo(CO)₂)₂ ″ ″ 0 — — 3-36 ((PPh₃)₂Co(CO)₂)₂ ″ ″ 0 — — 3-73(dmpe)₂Co 505  97 80 1.09 AG-Rep (PPh₃)₄Pd 150  ″ 82 220 1.05 AG-RepPd₂(DBA)₃ + 4 PEt₃ 150  ″ 81 254 1.06 AG-Rep (PEt₃)₂Pt(COD) 150  ″ 84236 1.04 3-61 (2,2′-bipyridyl)₂Fe 90 ″ 80 50 1.10 3-68 (PMe₃)₄Fe 50 ″ 0— — 3-69 (t-BuNC)₂FePhe₂ 90 ″ 0 — — 3-75 (PMe₃)₂FePhe₂ 50 ″ 96 84 1.113-75 (PMe₃)₂FePhe₂ 90 DMF 50 25 1.21 3-75 (2,2′-bipyridyl)FePhe₂ ″ ″ 9736 1.18 3-75 (t-BuNC)₂FePhe₂ ″ ″ 96 38 1.15 3-127 (PMe₃)₄Co 100  DMF 9722 1.11 3-124 (PMe₃)₄Co 50 THF 98 47 1.17 3-117 dmpeIrCl 25 THF 97 671.28 3-117 (PMe₃)IrCl 25 THF 96 89 1.14 3-112 (PEt₃)₂IrCl 50 THF 25 501.40 3-113 (CH₂(PEt₂)₂)₂IrC1 50 THF 97 122 1.21 3-114 (CH₂(PCy₂)₂)₂IrCl50 THF 94 188 1.17 3-118 dmpeRhCl 50 THF 98 99 1.16 3-118 (PMe₃)₂RhCl 50THF 39 238 1.22 3-103 dmpeCoPhe₂ 100  DMF 98 21 1.07

Example 5 Method of Preparing Oligo(Ethyleneglycol) Functionalized AminoAcids and Their Polymers: New Water Soluble Biocompatible Polypeptides

The following experiments describe the synthesis ofoligo(ethyleneglycol) functionalized lysine, serine, cysteine, andtyrosine NCA monomers, and their subsequent polymerization intooligo(ethyleneglycol) functionalized polypeptides.

General

Tetrahydrofuran (THF), hexane, N,N-dimethylformamide, and diethyl etherwere dried by passage through an alumina column under nitrogen prior touse. All reactions were conducted under an anhydrous nitrogenatmosphere, unless otherwise noted. The chemicals were purchased fromcommercial suppliers and used without purification. Co(PMe₃)₄ wasprepared according to the procedure of Klein, et al. [Klein, et al.,Methyltetrakis(trimethylphosphin)kobalt und seine Derivate, Chem. Ber.,108:944-955 (1975); Incorporated herein by reference]. The infraredspectra were recorded on a Perkin Elmer RX1 FTIR Spectrophotometercalibrated using polystyrene film. ¹H NMR spectra were recorded on aBruker AVANCE 200 MHZ spectrometer and were referenced to internalsolvent resonances. Tandem gel permeation chromatography/lightscattering (GPC/LS) was performed on a SSI pump equipped with a WyattDAWN DSP light scattering detector and Wyatt Optilab DSP. Separationswere effected by 10⁵ Å, 10⁴ Å, and 10³ Å Phenomenex 5 μm columns using0.1M LiBr in DMF as eluent at 60° C. Circular dichroism measurementswere carried out on a Olis Rapid Scanning Monochromator at roomtemperature. The path length of the quartz cell was 1.0 mm and theconcentration of peptide was 0.5 mg/mL. MALDITOF mass spectra werecollected using a Thermo BioAnalysis DYNAMO mass spectrometer running inpositive ion mode with samples prepared by mixing solutions of analytein THF with solutions of 2,5-dihydroxybenzoic acid in THF and allowingthe mixture to air dry.

N-Hydroxysuccinimidyl 2-[2-Methoxyethoxy)Ethoxy]Acetate, 1 Preparationof N_(α)-tert-butyloxycarbonyl-O-(2-(2-methoxyethoxy)ethyl)-L-serine,Compound 1, Scheme II

N_(α)-tert-butyloxycarbonyl-L-serine (4.59 g, 22.3 mmol) was dissolvedin N,N-dimethylformamide (100 mL), the solution was then cooled to 0° C.and treated with sodium hydride (1.97 g, 49.2 mmol).1-Bromo-2-(2-methoxyethoxy)ethane (10.0 g, 49.2 mmol) was added to thesolution and the reaction mixture was stirred at ambient temperature for3 h. The solvent was then removed under a reduced pressure at 40° C.bath temperature. The residue was dissolved in water (75 mL) and washedtwice with diethyl ether (30 mL each time). The aqueous layer was thenacidified to pH 3 with 1M HCl and then extracted with ethyl acetate. Theorganic layer was dried over anhydrous MgSO₄ and the solvent was removedin vacuo to yield the compound as a yellow oil (4.1 g, 63%). Thecompound has the following characteristics: FTIR (CHCl₃): 1735 (_(v)CO,s), 1712 (_(v)CO, s). ¹H NMR (CDCl₃): δ 5.63 (d,(CH₃)₃COC(O)NHCH(CH₂OCH₂CH₂OCH₂CH₂OCH₃)C(O)OH, 1H), 4.38 (s,(CH₃)₃COC(O)NHCH(CH₂OCH₂CH₂O—CH₂CH₂OCH₃)C(O)OH, 1H), 4.14-3.53 (m,(CH₃)₃COC(O)NHCH(CH ₂OCH₂CH₂O—CH ₂CH ₂OCH₃)C(O)OH, 10H), 3.47 (s,(CH₃)₃COC(O)NHCH(CH₂OCH₂CH₂OCH₂—CH₂OCH ₃)C(O)OH, 3H), 1.52 (s, (CH₃)₃COC(O)NHCH(CH₂O—CH₂CH₂OCH₂CH₂—OCH₃)C(O)OH, 9H). MALDITOF-MS: MH⁺:307.34 calcd, 309.19 found.

Preparation of O-(2-(2-methoxyethoxy)ethyl)-L-serine, Compound 2, SchemeII

The serine derivativeN_(α)-tert-butyloxycarbonyl-O-(2-(2-methoxy-ethoxy)ethyl)-L-serine wasused without further purification. The serine derivative (4.13 g, 16.9mmol) was dissolved in concentrated acetic acid (50 mL). After placingthe solution in an ice bath, 1M HCl (34 mL) was then added and themixture stirred for 30 min. The stirring was continued at ambienttemperature for 2 h and the solution was then concentrated under reducedpressure to yield a yellow oil. The oil was then neutralized with Et₃Nand the amine salt was removed by extraction with CH₃CN. The insolubleproduct was collected as a white solid (2.4 g, 58%). ¹H NMR (D₂O): δ3.87 (m, NH₂CH(CH ₂OCH₂CH₂OCH₂CH₂OCH₃)C(O)OH, 3H), 3.67-3.60 (m,NH₂CH(CH₂O—CH ₂CH ₂OCH ₂CH ₂OCH₃)C(O)OH, 8H), 3.34 (s,NH₂CH(CH₂OCH₂CH₂OCH₂CH₂O—CH ₃)C(O)OH, 3H). ¹³C {¹H} NMR (D₂O): δ 173.05(NH₂CH(CH₂OCH₂CH₂OCH₂—CH₂OCH₃)C(O)OH), 71.97, 70.87, 70.64, 70.48, 69.80(NH₂CH(CH₂OCH₂ CH₂OCH₂—CH₂OCH₃)C(O)OH), 59.10(NH₂CH(CH₂OCH₂CH₂OCH₂—CH₂OCH₃)C(O)OH)), 55.67(NH₂CH(CH₂OCH₂CH₂OCH₂—CH₂OCH₃)C(O)OH). MALDITOF-MS: MH⁺: 207.22 calcd,208.48 found. [α]_(D) ²³=−11.2 (c=0.05, H₂O).

Preparation of O-(2-(2-methoxyethoxy)ethyl)-L-serine NCA, Compound 3,Scheme II

To O-(2-(2-methoxyethoxy)ethyl)-L-serine (0.64 g, 2.6 mmol) was addedTHF (100 mL) and COCl₂ (1.63 mL of a 1.93M toluene solution) and themixture was stirred for 5 h at room temperature. The resulting solutionwas concentrated to give a yellow oil as the crude product (0.49 g,80%). The oil was crystallized from a tetrahydrofuran, toluene andhexane mixture (1:4:4) at −30° C. to give the product as a white solid(0.27 g, 45%). FTIR (THF): 1858 cm⁻¹ (υCO, s), 1792 cm⁻¹, (υCO, s), ¹HNMR (CDCl₃): δ 7.53 (s, RC(H)C(O)OC(O)NH, R=—CH₂OCH₂CH₂OCH₂CH₂OCH₃, 1H)4.40 (t, RC(H)C(O)OC(O)NH, R=—CH₂OCH₂CH₂OCH₂CH₂OCH₃ IH), 3.90 (d,RC(H)C(O)OC(O)NH, R=—CH ₂OCH₂CH₂OCH₂—CH₂OCH₃ 2H), 3.70-3.55 (m,RC(H)C(O)OC(O)NH, R=—CH₂OCH ₂CH ₂OCH ₂CH ₂OCH₃, 8H), 3.41 (s,RC(H)C(O)OC(O)NH, R=—CH₂OCH₂CH₂OCH₂—CH₂OCH ₃, 3H). ¹³C {¹H} NMR (CDCl₃):δ 169.82 (RC(H)C(O)OC(O)NH, R=—CH₂OCH₂CH₂OCH₂—CH₂OCH₃), 153.63(RC(H)C(O)OC(O)NH, R=—CH₂OCH₂CH₂OCH₂—CH₂OCH₃), 72.75, 72.26, 71.76,71.32, 71.07 (RC(H)C(O)OC(O)NH, R=—CH₂OCH₂ CH₂OCH₂—CH₂OCH₃) 59.95(RC(H)C(O)OC(O)NH, R=—CH₂OCH₂CH₂OCH₂—CH₂OCH₃), 59.78 (RC(H)C(O)OC(O)NH,R=—CH₂OCH₂CH₂OCH₂—CH₂OCH₃) [α]_(D) ²³=−37.6 (c=0.0 17, THF).

Preparation of Poly(O-(2-(2-methoxyethoxy)ethyl)-L-serine), Compound 4,Scheme II

O-(2-(2-methoxyethoxy)ethyl)-L-serine NCA (120 mg, 0.52 mmol) in DMF (2mL) was mixed with Co(PMe₃)₄ (3.8 mg, 0.010 mmol) in THF (0.5 mL) andstirred for 18 h. The polymer was precipitated from this solution byaddition to hexane (20 mL). The polymer dissolved in H₂O (5 mL) anddialyzed to remove impurities and then freeze-dried to give the productas a white solid (70 mg, 71%). FTIR (KBr): 1631 cm⁻¹ (amide I, s br),1523 cm⁻¹ (amide II, s br). ¹H NMR: δ 8.45 (d,—(NHCH(CH₂OCH₂CH₂OCH₂CH₂OCH₃)—C(O))_(n)—, IH), 4.61 (m,—(NHCH(CH₂OCH₂CH₂OCH₂H₂OCH₃)C(O))_(n)—, 1H), 3.80 (br s, —(NHCH(CH₂OCH₂CH₂OCH₂CH₂OCH₃)C(O))_(n)—, 2H), 3.67-3.61 (br m, —(NHCH(CH₂OCH ₂CH₂OCH ₂CH ₂OCH₃)C(O))_(n)—, 8H), 3.36 (s, —(NHCH(CH₂OCH₂CH₂OCH₂CH₂OCH₃)C(O))_(n)—, 3H). ¹³C {¹H} NMR: δ 174.23(—(NHCH(CH₂OCH₂CH₂OCH₂CH₂OCH₃)C(O))_(n)—), 72.06, 71.15, 70.75, 70.59(—(NHCH(CH₂OCH₂ CH₂OCH₂ CH₂OCH₃)C(O))_(n)—), 59.15(—(NHCH(CH₂OCH₂CH₂O—CH₂CH₂OCH₃)C(O))_(n)—, 54.52(—(NHCH(CH₂OCH₂CH₂OCH₂CH₂OCH₃)C(O))_(n)—). [α]_(D) ²³=−28.3 (c=0.012,H₂O).

Preparation of (2-(2-methoxyethoxy)ethyl)chloroformate, Compound 5,Scheme II

Di(ethyleneglycol) monomethyl ether (5.0 g, 42 mmol) in THF (30 ml) wasmixed with COC1₂ (32.3 ml of a 1.93M solution in toluene) at 0° C. andstirred for one hour. It was then stirred at 10° C. for additional twohours. The solvent and excess phosgene were removed under reducedpressure to yield clear oil (7.0 g, 92%). FTIR(CH₂Cl₂): 1780 cm⁻¹. Thiscompound was used without further purification.

Preparation of O-(2-(2-methoxyethoxy)ethyl)carbonyl-N^(a)Cbz-L-tyrosine,Compound 6, Scheme II

N_(α)-Cbz-L-tyrosine (5.00 g, 15.9 mmol) was dissolved in NaOH (0.634 g,15.9 mmol) in water (100 mL). (2-(2-methoxyethoxy)ethyl)chloroformate(4.34 g, 23.8 mmol) and 23.8 ml of 1M NaOH were added simultaneously at0° C. and the resulting mixture was stirred for one hour at thistemperature. The mixture was then stirred for an additional three hoursat ambient temperature. The solution was acidified with 1M HCl andextracted with ethyl acetate. The organic layer was dried over MgSO₄ andsolvent removed under reduced pressure to yield the product as a yellowoil (6.42 g, 91%). This compound was used without further purification.FTIR (CH₂Cl₂): 1764, 1725 cm⁻¹. ¹H NMR (CDCl₃): δ 3.39 (s, 3H),3.57-3.79 (m, 8H), 4.38 (m, 2H), 4.63 (s, 1H), 5.05 (s, 2H), 6.93-7.57(m, 9H).

Preparation of O-(2-(2-methoxyethoxy)ethyl)carbonyl-L-tyrosine NCA,Compound 7, Scheme II

O-(2-(2-methoxyethoxy)ethyl)carbonyl-N_(α)-Cbz-L-tyrosine (6.43 g, 14.3mmol) was dissolved in CH₂Cl₂ (150 ml) and (α,α-dichloromethyl methylether (2.46 g, 21.5 mmol) was added to the solution. The mixture wasthen refluxed for 40 h, and then the solvent was removed to yield theproduct as a yellow oil (3.42 g, 70.1%). FTIR (THF): 1790, 1855 cm⁻¹. ¹HNMR (CDCl₃): δ 7.04 (s, 4H), 4.53 (m, 1H), 4.31 (m, 2H), 3.71 (m, 2H),3.59 (m, 2H), 3.50 (m, 2H), 3.30 (s, 3H), 3.09-2.97 (m, 2H). ¹³C NMR(CDCl₃): δ 169.64, 153.73, 152.07, 150.55, 132.30, 130.74, 121.60,71.84, 70.52, 68.89, 67.92, 59.00, 58.81, 36.94.

Preparation of Poly(O-(2-(2-methoxyethoxy)ethyl)carbonyl-L-tyrosine),Compound 8, Scheme II

O-(2-(2-ethoxymethoxy)ethyl)carbonyl-L-tyrosine NCA (3.42 g, 10.0 mmol)was dissolved in THF (10 mL). Sodium t-butoxide (9.6 mg, 0.10 mmol) wasthen added. The resulting solution was stirred over night to give thepolymer as an off-white precipitate that was isolated by washing withdiethyl ether (50 mL) (1.94 g, 65%). FT-IR (THF): 1660, 1547 cm⁻¹.

Preparation of S-(2-(2-methoxyethoxy)ethyl)carbonyl-L-cysteine, Compound9, Scheme II

L-cysteine hydrochloride (2.00 g, 12.7 mmol) was dissolved in 1M aqueoussodium bicarbonate (25.4 ml) and the solution was diluted with water (75mL). The solution was cooled in an ice bath and then covered with ether(50 ml). (2-(2-methoxyethoxy)ethyl)chloroformate (2.31 g, 12.7 mmol) wasadded to the solution in one portion with vigorous stirring for one hourat 0° C. The temperature was allowed to rise to 10° C. and stirred foran additional two hours. The solvents were then removed under reducedpressure. The resulting solid was washed with methanol and the methanollayer was evaporated to yield the product as a white oil (2.21 g, 65%).FTIR: 1709 cm⁻¹. ¹H NMR (D₂O): δ 4.43 (m, 1IH), 4.24 (m, 2H), 3.83-3.47(m, 8H), 3.41 (s, 3H).

Preparation of S-(2-(2-methoxyethoxy)ethyl)carbonyl-L-cysteine NCA,Compound 10, Scheme II

S-(2-(2-ethoxymethoxy)ethoxy)carbonyl-L-cysteine (2.21 g, 8.26 mmol) wasmixed with THF (100 mL) and COCl₂ (5.13 ml of a 1.93 M solution intoluene). The mixture was stirred at ambient temperature for 5 hours andthe solvent was then removed under reduced pressure to yield the productas a yellow oil (1.94 g, 80.1%). FTIR: 1791, 1862 cm⁻¹.

Preparation of Poly(S-(2-(2-methoxyethoxy)ethyl)carbonyl-L-cysteine),Compound 11, Scheme II

O-(2-(2-ethoxymethoxy)ethoxy)carbonyl-L-cysteine NCA (1.94 g, 6.61 mmol)was dissolved in THF (10 mL) and sodium t-butoxide (6.4 mg, 0.067 mmol)was then added. The initially homogeneous solution was stirred overnight to give the polymer as an off-white precipitate that was isolatedby washing with diethyl ether (50 mL) (1.17 g, 70.9%). FTIR: 1635, 1517cm⁻¹.

A mixture of 2-[2-(2-methoxyethoxy)ethoxy]acetic acid (10 g, 58 mmol)and N-hydroxysuccinimide (7.5 g, 64 mmol) dissolved in THF (ca. 300 mL)in a round bottom flask was cooled using an ice water bath.Dicyclohexylcarbodiimide (12 g, 58 mmol) was then added with stirring. Awhite precipitate was observed to form after 5 min and the reactionmixture was then let stand in a refrigerator (4° C.) for 16 h. The whiteprecipitate, dicyclohexylurea, was removed by filtration and thefiltrate was concentrated under vacuum to give an oil. This crudeproduct was then dissolved in a small amount of THF (ca. 10 mL) and theresulting suspension was filtered to remove the precipitate. Thisprocedure was repeated until a clear solution was obtained upondissolution in THF. Removal of the residual THF under vacuum gave theproduct an oil (9.0 g, 59%). ¹H NMR (CDCl₃): δ 4.49 (s, —OC(O)CH ₂O—,2H), 3.77 (m, —OC(O)CH₂OCH ₂CH₂O—, 2H), 3.65 (m, —OCH₂CH ₂CH₂—, 4H),3.52 (m, —OCH₂CH ₂OCH₃, 2H), 3.34 (s, —CH₂OCH ₃, 3H), 2.82 (s, —C(O)CH₂CH ₂C(O)—, 4H).

N_(ε)-2-[2-(2-Methoxyethoxy)Ethoxy]Acetyl-N_(α)-CBZ-L-Lysine, Compound2, Scheme III

To a mixture of N_(α)-CBZ-L-Lysine (4.9 g, 17 mmol) and NaHCO₃ (2.0 g,23 mmol) in THF:H₂0 (75 mL:75 mL) was added 1 (3.2 g, 12 mmol) in THF(10 mL). After stirring for 1 h at 20° C., the THF was removed undervacuum. The product was extracted with ethyl acetate (2×50 mL), theorganic fractions were combined, and the solvent was removed undervacuum to leave a white solid. This crude product was recrystallizedfrom MeOH and diethyl ether to give 2 as white crystals (3.0 g, 59%).MP=115-117° C. ¹H NMR (CDCl₃): δ 7.25 (m, —CH₂C₆ H ₅, 5H), 5.18 (s, —CH₂C₆H₅, 2H), 4.65 (t, —NHCH(R)C(O)OH, 1H), 3.72 (m, —NHCH((CH₂)₃CH₂C(O)R)C(O)—+—O(CH ₂CH ₂O)₂CH ₃, 15H), 1.70 (m, —NHCH((CH₂)₃CH₂C(O)R)C(O)—, 6H).

N_(ε)-2-[2-(2-Methoxyethoxy)Ethoxy]Acetyl-L-Lysine-N-Carboxyanhydride,Compound 3, Scheme III

To a solution of 2 (4.9 g, 11 mmol) in anhydrous CH₂Cl₂ (125 mL) undernitrogen was added 1,1-dichlorodimethylether (1.5 mL, 17 mmol). Thesolution was then heated to reflux for 20 h, after which the solvent wasremoved under vacuum. The crude oil was crystallized from THF andhexanes to give 3 as white crystals (2.8 g, 75%). ¹H NMR (CDCl₃): δ 7.68(br s, —NH, 1H), 7.35 (br s, —NH, 1H), 4.30 (t, —NHCH(R)C(O)O—, 1H),3.15 (m, —NHCH((CH₂)₃CH ₂C(O)R)C(O)—+—O(CHCH ₂O)₂CH ₃, 15H), 1.70 (m,—NHCH((CH ₂)₃CH2C(O)R)C(O)—, 6H. FTIR(THF): 1856 cm⁻¹ (υCO, anhydrides), 1789 cm⁻¹ (υCO, anhydride, vs), 1677 cm⁻¹ (υCO, amide, s).

Poly(N_(ε)-2-[2-Methoxyethoxy)Ethoxy]Acetyl-L-Lysine), Compound 4.Scheme III

In the dry box, 3 (730 mg, 2.2 mmol) was dissolved in THF (15 mL) andplaced in a 75 mL reaction tube which could be sealed with a Teflonstopper. An aliquot of 2,2′-bipyridyl)Ni(1,5-cyclooctadiene) (600 μL ofa 36 mM solution THF) was then added via syringe to the flask. A stirbarwas added and the flask was sealed, removed from the dry box, andstirred in a thermostated 25° C. bath for 24 h. Polymer was isolated byaddition of the reaction mixture to diethyl ether causing precipitationof the polymer. The polymer was then dissolved in THF and reprecipitatedby addition to diethyl ether. The polymer was dried in vacuo to give 4as a white fibrous solid (550 mg, 87% yield). GPC of the polymer in 0.1MLiBr in DMF at 60° C.: M_(n)=101,000; M_(w)/M_(n)=1.21. FTIR(THF): 1672cm⁻¹ (υCO, amide, s), 1650 cm⁻¹ (υCO, Amide I, br vs), 1538 cm⁻¹ (υCO,Amide II, br s).

Example 6 Synthesis of Self-Assembling Amphiphilic Block Copolypeptidesfor Biomedical Applications

In the examples below, we prepared di- and tri-block copolypeptides ofthe general architectures: (EG-Lys)_(x)-(insoluble block)_(y) and(EG-Lys)_(x)-(insoluble block)_(y)-(EG-Lys)_(z), where x, y, and zrepresent the number of amino acids in each domain.

Sample Synthesis of aPoly(N_(ε)-2-[2-(2-Methoxyethoxy)ethoxy]acetyl-L-Lysine)-block-(L-Leucine/L-Valine)diblock copolymer

In the dry-box, 100 mg ofN_(ε)-2-[2-(2-Methoxyethoxy)ethoxy]acetyl-L-Lysine)-N-carboxyanhydride,EG-Lys NCA, was dissolved in anhydrous THF (3 mL) and placed in a 15 mLreaction tube, which could be sealed with a Teflon stopper. Into thereaction tube, which contained a stirbar, an aliquot of (PMe₃)₄)Co (2.37mg in 1 ml of THF) was added. The flask was sealed and stirred overnightto form the poly(EG-Lys) block. A mixture ofL-leucine-N-carboxyanhydride, Leu NCA, (5 mg) andL-valine-N-carboxyanhydride, Val NCA, (1 mg) was dissolved in anhydrousTHF (1 mL) and then added to the reaction flask. After stirring for anadditional 16 h, the block copolymer was isolated and purified byremoving the solvent from the reaction mixture in vacuo followed byresuspension of the residue in double distilled water and dialysis ofthis solution against 4 liters of double distilled water for 8 h using aSpectrapore dialysis membrane with a molecular weight cut-off of 1000.The dialysis was repeated twice and the copolymer was isolated byfreeze-drying of the solution (yield: 76 mg). GPC analysis of thepolymer: M_(n)=39,000 and M_(w)/M_(n)=1.2. FTIR(THF): 1650 cm⁻¹ (υCO,amide I, vs), 1540 cm⁻¹ (υCO, amide II, s).

Sample Synthesis of aPoly(N_(ε)-2-[2-(2-Methoxyethoxy)ethoxy]acetyl-L-Lysine)-block-(L-Leucine/L-Valine)-block-(N_(ε)-2-[2-(2-Methoxyethoxy)ethoxy]acetyl-L-Lysinetriblock copolymer

In the dry-box, 50 mg ofN_(ε)-2[2-(2-Methoxyethoxy)ethoxy]acetyl-L-Lysine)-N-carboxyanhydride,EG-Lys NCA, was dissolved in anhydrous THF (3 mL) and placed in a 15 mLreaction tube, which could be sealed with a Teflon stopper. Into thereaction tube, which contained a stirbar, an aliquot of (PMe₃)₄)Co (2.37mg in 1 ml of THF) was added. The flask was sealed and stirred overnightto form the poly(EG-Lys) block. A mixture ofL-leucine-N-carboxyanhydride, Leu NCA, (5 mg) andL-valine-N-carboxyanhydride, Val NCA, (1 mg) was dissolved in anhydrousTHF (1 mL) and then added to the reaction flask to form the centralblock. After stirring for an additional 16 h, 50 mg of EG-Lys NCA wasdissolved in THF (1 mL) and added to the reaction tube, which wasstirred for an additional 16 h to form the final poly(EG-Lys) block ofthe complete triblock copolymer. The copolymer was isolated and purifiedby removing the solvent from the reaction mixture in vacuo followed byresuspension of the residue in double distilled water and dialysis ofthis solution against 4 liters of double distilled water for 8 h using aSpectrapore dialysis membrane with a molecular weight cut-off of 1000.The dialysis was repeated twice and the copolymer was isolated byfreeze-drying of the solution (yield: 78 mg). GPC analysis of thepolymer: M_(n)=41,000 and M_(w)/M_(n)=1.1. FTIR(THF): 1650 cm⁻¹ (υCO,amide I, vs), 1540 cm⁻¹ (υCO, amide II, s).

Vesicle Formation

Spontaneous vesicle formation from di- and tri-block copolypeptides wasobserved under a light microscope after initial dissolution in waterwhich gave milky suspensions. The size of the vesicles varied widely,with diameters up to several microns. Smaller vesicles were prepared bysonication of the solutions of large vesicles, similar to the way lipidvesicles are manipulated. Polymers were typically dissolved in doublydistilled water at concentrations of 5 mg/mL and then sonicated for 9min at 13 watts of power using a Via Cell sonicator. The sonication wasrepeated 3 times, after which the opalescent suspensions became clearsolutions.

Vesicle Characterization

Light Microscopy was performed on a Nikon Optiphot2-POL. Initial sampleswere approximately 5 mg/ml in doubly distilled water and 50μ of samplewas deposited on a glass slide and topped with a cover-slip forvisualization.

Light scattering was performed on a Brookhaven Instruments Dynamic LightScattering (DLS) system, with a laser power of 30 mwatts and wavelengthof 546 nm. Samples were sonicated as indicated above and added to DLScuvettes. The sonicated vesicles had average diameters ranging from 50nm to 500 nm, depending on copolymer chain length and amino acidcomposition (e.g., see Table 9 below).

TABLE 9 Name* Composition Molecular Weight Vesicle Diameter PLV10 90% P,10% LV 10 kDa 132 nm PLV10 90% P, 10% LV 22 kDa 147 nm PLV10 90% P, 10%LV 40 kDa 197 nm PLV10 90% P, 10% LV 90 kDa 230 nm PLV20 80% P, 20% LV25 kDa 175 nm PLV30 70% P, 30% LV 30 kDa 207 nm PLV40 60% P, 40% LV 35kDa 217 nm *Key: P = poly (EG₂-L lysine) domain; LV = hydophobic domaincomposed of a random copolymer of L-leucine (L) and L-valine (V) with aninternal composition of 75% leucine and 25% valine. Thus, PLV10 is adiblock copolymer where the P block makes up 90 mole percent of thetotal polymer size and the LV block makes up 10% of the total copolymer.

Synthesis of a Poly (ε-benzyloxycarbonyl-L-Lysine-block-L-Leucine)diblock copolymer

In the dry box, ε-benzyloxycarbonyl-L-lysine NCA (hereinafter, Lys NCA)(100 mg, 0.33 mmol) is dissolved in THF (2.0 mL) and placed in a 15 mLreaction tube which can be sealed with a Teflon stopper. An aliquot of(PMe₃)₄Co (237 mL of a 10 mg/mL solution in THF) is added via syringe tothe Lys NCA solution. The flask is then sealed and allowed to stir for 8hours. At the end of the 8 hour period a small aliquot of solution isremoved for GPC/LS analysis (M_(n)=55,000; M_(w)/M_(n)=1.20). A solutionof L-leucine NCA is prepared in THF (concentration=10 mg/mL) and analiquot (0.58 mL) is transferred via syringe into the polymerizationmixture. The reaction is left to stir for another 3 hour period. Afterthis time, an aliquot of solution is removed for FTIR analysis toconfirm the complete consumption of NCA monomer (as determined bymeasuring characteristic NCA absorptions at 1854 cm⁻¹ and 1790 cm⁻¹) andproduction of polymer (as determined by measuring characteristicpolypeptide absorptions at ca. 1650 cm⁻¹ and 1540 cm⁻¹). The reactionmixture is then removed from the dry box and the THF is evaporated usinga gentle stream of dry nitrogen to give the crude polymer as a hardfilm.

Synthesis of a Poly(L-Lysine-block-L-Leucine) Diblock Copolymer

The crude polymer from above, in the original reaction tube, isdissolved in trifluoroacetic acid (TFA, 3.0 mL) and the tube is thenplaced in an ice bath where hydrobromic acid is added (220 mL of a 33 wt% HBr in glacial acetic acid solution, 4 equivalents). The solution iscapped with a Teflon stopper and stirred vigorously for 1 hour. Thepolymer is then precipitated out of solution by the addition of 10 mL ofdiethyl ether. The suspension is centrifuged and the supernatantdiscarded. The sample is washed twice more with diethyl ether (10 mLeach time). The polymer is then dried with a nitrogen stream anddissolved in deionized water (10 mL). The resulting solution is placedin a dialysis bag (molecular weight cut off of ca. 1000) and dialyzedagainst deionized water in a 5 liter container. The dialysis water isexchanged four times at eight hour intervals. The resulting polymersolution is then frozen with liquid nitrogen and then freeze-dried togive the dry polymer (63 mg, 87% yield).

Synthesis of aPoly((y-benzyl-L-glutamate)-block-Poly((E-benzyloxycarbonyl-L-lysine)-block-(L-Leucine/L-Valine))Block Copolymer

In the dry box, g-benzyl-L-glutamate NCA (hereinafter, Glu NCA) (215 mg,0.82 mmol) is dissolved in THF (4.0 mL) and placed in a 20 mL glass vialwhich could be sealed with a plastic cap. An aliquot of (PMe₃)₄Co (1.0mL of a 6.6 mg/mL solution in THF) is added via syringe to the Glu NCAsolution. The flask is then sealed and allowed to stir for 8 hours. LysNCA (250 mg, 0.82 mmol) is then weighed out and dissolved in THF (5.0mL). This solution is then injected into the polymerization reaction,which is capped and left to stir for 8 hours. A 75/25 molar ratiomixture of L-Leucine NCA and L-Valine NCA (100 mg L-Leucine and 30 mgL-Valine) is dissolved in THF (1.3 mL) and an aliquot of this solution(280 mL) is transferred via syringe into the reaction flask. Thereaction is left to stir for another 8-hour period. After this time, analiquot of solution is removed for FTIR analysis to confirm the completeconsumption of NCA monomer (as determined by measuring characteristicNCA absorptions at 1854 cm⁻¹ and 1790 cm⁻¹) and production of polymer(as determined by measuring characteristic polypeptide absorptions atca. 1650 cm⁻¹ and 1540 cm⁻¹). The reaction mixture is then removed fromthe dry box and the THF is evaporated using a gentle stream of drynitrogen to give the crude polymer as a hard film.

Synthesis of a Poly(L-GlutamicAcid-block-L-Lysine-block-(L-Leucine/L-Valine)) Block Copolymer

The crude polymer from above, in the original reaction tube, isdissolved in trifluoroacetic acid (TFA, 3.0 mL) and the tube is thenplaced in an ice bath where hydrobromic acid is added (220 mL of a 33 wt% HBr in glacial acetic acid solution, 4 equivalents). The solution iscapped with a Teflon stopper and stirred vigorously for 1 hour. Thepolymer is then precipitated out of solution by the addition of 10 mL ofdiethyl ether. The suspension is centrifuged and the supernatantdiscarded. The sample is washed twice more with diethyl ether (10 mLeach time). The polymer is then dried with a nitrogen stream anddissolved in deionized water (10 mL). The resulting solution is placedin a dialysis bag (molecular weight cut off of ca. 1000) and dialyzedagainst deionized water in a 5 liter container. The dialysis water isexchanged four times at eight hour intervals. The resulting polymersolution is then frozen with liquid nitrogen and then freeze-dried togive the dry polymer: 155 mg (67% yield).

Synthesis of aPoly((e-benzyloxycarbonyl-L-lysine)-block-(Poly((y-benzyl-L-glutamate)-block-(L-Leucine/L-Valine))Block Copolymer

In the dry box, Lys NCA (250 mg, 0.82 mmol) is dissolved in THF (5.0 mL)and placed in a 20 mL glass vial which can be sealed with a plastic cap.An aliquot of (PMe₃)₄Co (1.0 mL of a 6.6 mg/mL solution in THF) is addedvia syringe to the Lys NCA solution. The flask is then sealed andallowed to stir for 8 hours. Glu NCA (215 mg, 0.817 mmol) is weighed outand then dissolved in THF (4.0 mL). This solution is then injected intothe polymerization reaction, which is capped and left to stir for 8hours. A 75/25 molar ratio mixture of L-Leucine NCA and L-Valine NCA(100 mg L-Leucine and 30 mg L-Valine) is dissolved in THF (1.3 mL) andan aliquot of this solution (280 mL) is transferred via syringe into thereaction flask. The reaction is left to stir for another 8-hour period.After this time, an aliquot of solution is removed for FTIR analysis toconfirm the complete consumption of NCA monomer (as determined bymeasuring characteristic NCA absorptions at 1854 cm⁻¹ and 1790 cm⁻¹) andproduction of polymer (as determined by measuring characteristicpolypeptide absorptions at ca. 1650 cm⁻¹ and 1540 cm⁻¹). The reactionmixture is then removed from the dry box and the THF is evaporated usinga gentle stream of dry nitrogen to give the crude polymer as a hardfilm.

Synthesis of a Poly(L-Lysine-block-L-GlutamicAcid-block-(L-Leucine/L-Valine)) Block Copolymer

The crude polymer from above, in the original reaction tube, isdissolved in trifluoroacetic acid (TFA, 3.0 mL and the tube is thenplaced in an ice bath where hydrobromic acid is added (220 mL of a 33 wt% HBr in glacial acetic acid solution, 4 equivalents). The solution iscapped with a Teflon stopper and stirred vigorously for 1 hour. Thepolymer is then precipitated out of solution by the addition of 10 mL ofdiethyl ether. The suspension is centrifuged and the supernatantdiscarded. The sample is washed twice more with diethyl ether (10 mLeach time). The polymer is then dried with a nitrogen stream anddissolved in deionized water (10 mL). The resulting solution is placedin a dialysis bag (molecular weight cut off of ca. 1000) and dialyzedagainst deionized water in a 5 liter container. The dialysis water isexchanged four times at eight hour intervals. The resulting polymersolution is then frozen with liquid nitrogen and then freeze-dried togive the dry polymer: 182 mg (79% yield).

Synthesis of aPoly((g-benzyl-L-glutamate/e-benzy[oxycarbonyl-L-lysine)-block-(L-Leucine[L-Valine))Block Copolymer

In the dry box, Glu NCA (215 mg, 0.82 mmol) and Lys NCA (250 mg, 0.82mmol) are mixed together and dissolved in THF (9.0 mL and then placed ina 20 mL glass vial which could be sealed with a plastic cap. An aliquotof (PMe3)4CO (1.0 mL of a 6.6 mg/mL solution in THF) is added viasyringe to the NCA solution. The flask is then sealed and allowed tostir for 8 hours. A 75/25 molar ratio mixture of L-Leucine NCA andL-Valine NCA (100 mg L-Leucine and 30 mg L-Valine) is dissolved in THF(1.3 mL) and an aliquot of this solution (280 mL) is transferred viasyringe into the reaction flask. The reaction is left to stir foranother 8-hour period. After this time, an aliquot of solution isremoved for FTIR analysis to confirm the complete consumption of NCAmonomer (as determined by measuring characteristic NCA absorptions at1854 cm⁻¹ and 1790 cm⁻¹) and production of polymer (as determined bymeasuring characteristic polypeptide absorptions at ca. 1650 cm⁻¹ and1540 cm⁻¹). The reaction mixture is then removed from the dry box andthe THF is evaporated using, a gentle stream of dry nitrogen to give thecrude polymer as a hard film.

Synthesis of a Poly((L-GlutamicAcid/L-Lysine)-block-(L-Leucine/L-Valine)) Block Copolymer

The crude polymer from above, in the original reaction tube, isdissolved in trifluoroacetic acid (TFA, 3.0 mL) and the tube is thenplaced in an ice bath where hydrobromic acid is added (220 mL of a 33 wt% HBr in glacial acetic acid solution, 4 equivalents). The solution iscapped with a Teflon stopper and stirred vigorously for 1 hour. Thepolymer is then precipitated out of solution by the addition of 10 mL ofdiethyl ether. The suspension is centrifuged and the supernatentdiscarded. The sample is washed twice more with diethyl ether (10 mLeach time). The polymer is then dried with a nitrogen stream anddissolved in deionized water (10 mL). The resulting solution is placedin a dialysis bag (molecular weight cut off of ca. 1000) and dialyzedagainst deionized water in a 5 liter container. The dialysis water isexchanged four times at eight hour intervals. The resulting polymersolution is then frozen with liquid nitrogen and then freeze-dried togive the dry polymer: 174 mg (75% yield).

Synthesis of a Poly ((g-benzyl-L-glutamate)-block-(L-Leucine/L-Valine))Block Copolymer

In the dry box, Glu NCA (250 mg, 0.95 mmol) is dissolved in THF (5.0 mL)and placed in a 15 mL reaction tube which could be sealed with a Teflonstopper. An aliquot of (PMe3)₄Co (580 μL of a 6.6 mg/mL solution in THF)is added via syringe to the Glu NCA solution. The flask is then sealedand allowed to stir for 8 hours. A 75/25 molar ratio mixture ofL-Leucine NCA and L-Valine NCA (100 mg Leucine and 30 mg L-Valine) isdissolved in THF (1.3 mL) and an aliquot of this solution (160 mL) istransferred via syringe into the reaction flask. The reaction is left tostir for another 3-hour period. After this time, an aliquot of solutionis removed for FTIR analysis to confirm the complete consumption of NCAmonomer (as determined by measuring characteristic NCA absorptions at1854 cm⁻¹ and 1790 cm⁻¹) and production of polymer (as determined bymeasuring characteristic polypeptide absorptions at ca. 1650 cm⁻¹ and1540 cm⁻¹). The reaction mixture is then removed from the dry box andthe THF is evaporated using a gentle stream of dry nitrogen to give thecrude polymer as a hard film.

Synthesis of a Poly(L-Glutamic Acid-block-(L-Leucine/L-Valine)) BlockCopolymer

The crude polymer from above, in the original reaction tube, isdissolved in trifluoroacetic acid (TFA, 3.0 mL) and the tube is thenplaced in an ice bath where hydrobromic acid is added (220 mL of a 33 wt% HBr in glacial acetic acid solution, 4 equivalents). The solution iscapped with a Teflon stopper and stirred vigorously for 1 hour. Thepolymer is then precipitated out of solution by the addition of 10 mL ofdiethyl ether. The suspension is centrifuged and the supernatantdiscarded. The sample is washed twice more with diethyl ether (10 mLeach time). The polymer is then dried with a nitrogen stream anddissolved in deionized water (10 mL). The resulting solution is placedin a dialysis bag (molecular weight cut off of ca. 1000) and dialyzedagainst deionized water in a 5 liter container. The dialysis water isexchanged four times at eight hour intervals. The resulting polymersolution is then frozen with liquid nitrogen and then freeze-dried togive the dry polymer: 96 mg (72% yield).

Synthesis of aPoly(e-benzyloxycarbonyl-L-Lysine)-block-(L-Leucine/L-Valine) BlockCopolymer

In the dry box, Lys NCA (1.50 g, 4.90 mmol) is dissolved in THF (30.0mL) and placed in a 125 mL flask which could be sealed with a glassstopper. An aliquot of (PMe₃)₄Co (1.30 mL of a 30 mg/mL solution in THF)is added via syringe to the Lys NCA solution. The flask is then sealedand allowed to stir for 8 hours. A 75/25 molar ratio mixture ofL-Leucine NCA and L-Valine NCA (70 mg L-Leucine and 21 mg L-Valine) isdissolved in THF (1.0 mL) and this solution is transferred into thereaction flask. The reaction is left to stir for another 3-hour period.After this time, an aliquot of solution is removed for FTIR analysis toconfirm the complete consumption of NCA monomer (as determined bymeasuring characteristic NCA absorptions at 1854 cm⁻¹ and 1790 cm⁻¹) andproduction of polymer (as determined by measuring characteristicpolypeptide absorptions at ca. 1650 cm⁻¹ and 1540 cm⁻¹). The reactionmixture is then removed from the dry box and the THF is evaporated usinga gentle stream of dry nitrogen to give the crude polymer as a hardfilm.

Synthesis of a Poly(L-Lysine)-block-(L-Leucine/L-Valine) Block Copolymer

The crude polymer from above, in the original reaction tube, isdissolved in trifluoroacetic acid (TFA, 20 mL) and the tube is thenplaced in an ice bath where hydrobromic acid is added (3.4 mL of a 33 wt% HBr in glacial acetic acid solution, 4 equivalents). The solution iscapped with a Teflon stopper and stirred vigorously for 1 hour. Thepolymer is then precipitated out of solution by the addition of 50 mL ofdiethyl ether. The suspension is centrifuged and the supernatantdiscarded. The sample is washed twice more with diethyl ether (50 mLeach time). The polymer is then dried with a nitrogen stream anddissolved in deionized water (150 mL). The resulting solution is placedin a dialysis bag (molecular weight cut off of ca. 1000) and dialyzedagainst deionized water in a 5 liter container. The dialysis water isexchanged four times at eight hour intervals. The resulting polymersolution is then frozen with liquid nitrogen and then freeze-dried togive the dry polymer (K₉₀(L₇₅N₂₅)₁₀): 1.02 g (94% yield).

Preparation of Spherical Vesicles from K₉₀(L75/V₂₅)₁₀ Polymers

Dry polymer is taken from the freeze dryer as described previously anddissolved in concentrated salt solution (2M NaCl solution at 8 mg/mL). Asmall volume of solution (2.0 mL) is placed in one of the wells of a 24well plate and sonicated vigorously with a microcell sonicating probe.The power supply is set at 11 Watts and 5 mins. The solution is allowedto cool and the 11 Watt 5 min sonication is repeated once more.Following this, the solution is noticeably turbid and had a high densityof ˜1 mm sized hollow, spherical particles visible under a NikonOptiphot2-POL light microscope. The vesicles are stable in solutionfor >2 weeks.

Preparation of Hydrogels from K₉₀(L₇₅/V₂₅)₁₀ Polymers

Solid, freeze-dried polymer (20 mg) is dissolved in deionized water (1.0mL) and vigorously agitated using a mechanical stirrer until the polymeris dispersed. At this point, the mixture behaved as an extremely viscousgel. These water based gels can be formed by directly dispersing solidK₉₀(L₇₅/V₂₅)₁₀ in water at concentrations>0.5% w/v.

Example 6 Ruthenium Initiators for Synthesis of Polypeptides and BlockCopolypeptides

General.

Infrared spectra were recorded on a Perkin Elmer 1605 FTIRSpectrophotometer calibrated using polystyrene film. Tandem gelpermeation chromatography/light scattering (GPC/LS) was performed on aSpectra Physics Isochrom liquid chromatograph pump equipped with a WyattDAWN DSP light scattering detector and Wyatt Optilab DSP. Separationswere effected by 10⁵ Å and 10³ Å Phenomenex 5μ columns using 0.1M LiBrin DMF at 60° C. as eluent. NMR spectra were measured on a Bruker AMX500 MHz spectrometer. Chemicals were obtained from commercial suppliersand used without purification unless otherwise stated. Compound 1 (seeEq. 9) was prepared according to the literature procedure (Yamakawa, M.;Ito, H.; Noyori, R. J. Am. Chem. Soc., 2000, 122, 1466-1478;incorporated herein by reference). Hexanes, THF, and THF-d₈ werepurified by distillation from sodium benzophenone ketyl. DMF and DMF-d₇were purified by drying over 4 Å molecular sieves followed by vacuumdistillation.

Sample Polymerization of Glu-NCA with 1+3 PMe³.

In the dry box, Glu NCA (50 mg, 0.2 mmol) was dissolved in DMF (0.5 mL)and placed in a 25 mL reaction tube which could be sealed with a Teflonstopcock. An aliquot of 1+3 PMe₃ (25 μL of a 40 mM solution in DMF) wasthen added via syringe to the flask. A stirbar was added and the flaskwas sealed, removed from the dry box, and placed in a thermostated 25°C. bath for 16 h. Polymer was isolated by addition of the reactionmixture to methanol containing HCl (1 mM) causing precipitation of thepolymer. The polymer was then dissolved in THF and reprecipitated byaddition to methanol. The polymer was dried in vacuo to give a whitestringy solid, PBLG (39 mg, 93% yield). ¹³C {¹H} NMR, ¹H NMR, and FTIRspectra of this material were identical to data found for authenticsamples of PBLG. GPC of the polymer in 0.1M LiBr in DMF at 60° C.:M_(n)=12,000; M_(w)/M_(n)=1.18.

Although the present invention has been described in considerable detailwith reference to certain preferred versions thereof, other versions arepossible. For example, the lengths of each domain, and composition ofthe insoluble block domains, can be varied during the synthesis tomodify the self-assembled structures that are formed. In addition, D- orL- or stereomixtures of amino acids can be used in these blockcopolymers to modify polypeptide secondary structure or to modifybiological stability and interactions. Therefore, the spirit and scopeof the present invention should not be limited to the description of thepreferred versions contained herein.

1-20. (canceled)
 21. A five membered amido-containing metallacyclecomprising a molecule of the general formula:

wherein M is a low valent transition metal; L is a Lewis Base ligand;R1, R2 and R3 comprises a side chain of an amino acid selected from thegroup consisting of alanine, arginine, asparagine, aspartic acid,cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine and valine; and R4 is a hydrogen moiety or apolyaminoacid chain.
 22. A six membered amido-containing metallacyclecomprising a molecule of the general formula:

wherein M is a low valent transition metal; L is a Lewis Base ligand;R1, R2, R3, R5 and R6 is a side chain of an amino acid selected from thegroup consisting of alanine, arginine, asparagine, aspartic acid,cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine and valine; and R4 is a polyaminoacid chain. 23.The composition of claim 21 wherein the metal is a transition metalselected from the group consisting of nickel, palladium, platinum,cobalt, rhodium, iridium and iron.
 24. The composition of claim 21wherein the Lewis Base ligand is selected from the group consisting ofpyridyl ligands, diimine ligands, bisoxazoline ligands, alkyl phosphineligands, aryl phosphine ligands, tertiary amine ligands, isocyanideligands and cyanide ligands.
 25. A five membered amido-containingmetallacycle comprising a

molecule of the general formula: wherein M is a low valent transitionmetal; L is a Lewis Base ligand; one of R1 and R2 is an amino acid sidegroup and the other is hydrogen; and R3 is any functional end groupcapable of being attached to a primary amine group.
 26. Theamido-containing metallacycle of claim 25, wherein R1 or R2 comprises aside chain of an amino acid selected from the group consisting ofalanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid,glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine andvaline.
 27. The amido-containing metallacycle of claim 25, wherein R1 orR2 comprises a side chain of an amino acid selected from the groupconsisting of oligo(ethyleneglycol) functionalized (EG-)cysteine,EG-lysine, EG-serine, and EG-tyrosine.
 28. The amido-containingmetallacycle of claim 25, wherein R3 is a peptide, oligosaccharide,oligonucleotide, fluorescent molecule, polymer chain, small moleculetherapeutic, or chemical linker that couples the polypeptide to anothermolecule.
 29. A method of making an amphiphilic block copolypeptide,comprising the steps of: (1) generating a soluble block polypeptide bycombining an amount of an oligo (ethyleneglycol) functionalizedaminoacid-N-carboxyanhydride (EG-aa-NCA) monomer with an initiatormolecule; and (2) attaching an insoluble block by combining the solubleblock with a composition comprising at least one other amino acid NCAmonomer.
 30. The method of claim 29, wherein the amino acid component ofthe EG-aa-NCA monomer is lysine, serine, cysteine, or tyrosine.
 31. Themethod of 29 wherein the insoluble block contains a mixture of aminoacids.
 32. A method of adding an aminoacid-N-carboxyanhydride (NCA)monomer to a soluble block polypeptide, comprising combining the NCAmonomer with the soluble block polypeptide, said soluble block havingone or more oligo(ethyleneglycol)-functionalized amino acid residues, sothat the NCA monomer is added to the polypeptide.
 33. A polypeptidecomposition comprising a block copolypeptide having: (a) a total numberof overall monomer units that is greater than about 100 amino acidresidues; and (b) a distribution of chain-lengths of at least about1.01<Mw/Mn<1.25.
 34. The block copolypeptide of claim 33, wherein saidpolypeptide has a number of overall monomer units that are greater thanabout 2.50 amino acid residues.
 35. The block copolypeptide of claim 33,wherein said polypeptide comprises a least 3 blocks of consecutiveidentical amino acid monomer units.
 36. The block copolypeptide of claim33, wherein at least one of the blocks is componentsγ-benzyl-L-glutamate.
 37. The block copolypeptide of claim 33, whereinat least one of the blocks is components E-carbobenzyloxy-L-lysine. 38.The block copolypeptide of claim 33, wherein said polypeptide iscomposed of amino acid components γ-benzyl-L-glutamate andε-carbobenzyloxy-L-lysine.
 39. The block copolypeptide of claim 33,wherein said polypeptide is selected from the group consisting of apoly(ε-benzyloxycarbonyl-L-Lysine-block-γ-benzyl-L-glutamate),PZLL-b-PBLG, diblock copolymer and apoly(γ-benzyl-L-glutamate-block-ε-benzyloxycarbonyl-L-Lysine-block-γ-benzyl-L-glutamate)triblock copolymer.
 40. An amphiphilic block copolypeptide comprising asoluble block polypeptide having one or moreoligo(ethyleneglycol)-conjugated amino acid residues and an insolubleblock comprised substantially of nonionic amino acid residues.
 41. Anamphiphilic block copolypeptide comprising: (1) a soluble blockpolypeptide having EG-lysine residues, and (2) an insoluble blockpolypeptide containing a mixture of two to three different kinds ofamino acid components in a statistically random sequence.
 42. Anamphiphilic block copolypeptide consisting of at least 3 blocks, whereinone or more of the blocks is a soluble block polypeptide and anotherblock is an insoluble block polypeptide.
 43. An amphiphilic blockcopolypeptide comprising a soluble block polypeptide and an insolubleblock polypeptide, said soluble block having at least about 30% molepercent identical amino acid residues having charged oroligo(ethyleneglycol)-conjugated side chains and said insoluble blockcomprising at about 60 to 100 mole percent nonionic amino acid residues.44. The amphiphilic block copolypeptide of claim 43 wherein theinsoluble block comprises about 3 to about 60 mole percent of the totalcopolypeptide.
 45. The amphiphilic block copolypeptide of claim 43wherein the nonionic amino acid residues are selected from the groupconsisting of phenylalanine, leucine, valine, isoleucine, alanine andmethionine.
 46. The amphiphilic block copolypeptide of claim 43 whereinthe amino acid residues having charged side chains are selected from thegroup consisting of glutamic acid, aspartic acid, arginine, histidine,lysine, and ornithine.
 47. The amphiphilic block copolypeptide of claim43 wherein the amino acid residues havingoligo(ethyleneglycol)-conjugated side chains are selected from the groupconsisting of EG-cysteine, EG-lysine, EG-serine, and EG-tyrosine.
 48. Achain-end functionalized block polypeptide having ten or moreconsecutive identical amino acid residues and an endgroup selected fromthe group consisting of an oligosaccharide, oligonucleotide, fluorescentmolecule, polymer chain, small molecule therapeutic, or reactivechemical linker to attach the block copolypeptide to another molecule.49. A chain-end functionalized block copolypeptide having an end groupselected from the group consisting of a napthyl group, an alkyl group,an allyl group, and cysteinamide.
 50. A polyaminoacid chain comprisingat least ten consecutive oligo(ethyleneglycol)-conjugated amino acidresidues.
 51. A method of forming vesicles comprising the step ofsuspending the amphiphilic block copolypeptides of claim 40 an aqueoussolution so that the copolypeptides spontaneously self assemble intovesicles.
 52. The method of claim 51, further comprising the stepsonicating the suspended vesicles to form smaller vesicles having adiameter of about 50 nm to about 500 nm.
 53. Vesicle-containingcompositions comprising the amphiphilic block copolypeptides of claim 40and water.
 54. A method for making EG-functionalized amino acidmonomers, comprising the step of combining an ethyleneglycol (EG)derivative with an amino acid having a reactive side group.
 55. Themethod of claim 54, wherein the EG derivative has the general formula(CH₃OCH₂CH₂)_(n)X; wherein n is about 1 to 3, and X is a reactive groupselected from the group consisting of chloroformate,N-hydroxysuccidimydyl acetate, and halide.
 56. The method of claim 54,wherein the amino acid is selected from the group consisting of lysine,serine, cysteine, and tyrosine.
 57. The method of claim 54, furthercomprising the step of converting the EG functionalized amino acid to anNCA monomers.
 58. A method of making a soluble block polypeptidecomprising the step of combining an amount of an EG functionalizedaminoacid-N-carboxyanhydride (NCA) monomer with an initiator moleculecomprising a low valent transition metal-Lewis Base ligand complex sothat a EG functionalized polyaminoacid chain is generated.
 59. Themethod of claim 58 wherein the low valent transition metal is selectedfrom the group consisting of nickel, palladium, platinum, cobalt,rhodium, iridium and iron.
 60. The method of claim 58 wherein the LewisBase ligand is selected from the group consisting of pyridyl ligands,diimine ligands, bisoxazoline ligands, alkyl phosphine ligands, arylphosphine ligands, tertiary amine ligands, isocyanide ligands andcyanide ligands.
 61. The method of claim 58 wherein the EGfunctionalized-aminoacid-N-carboxyanhydride monomer is selected from thegroup consisting of EG-cysteine, EG-lysine, EG-serine, and EG-tyrosine.62. A method of preparing a polypeptide having a defined end groupcomprising the steps of: a) combining an alloc-amino acid amide with aninitiator comprising a low valent transition metal-Lewis Base ligandcomplex for a time and under conditions effective to form anamido-amidate metallacycle; and b) adding one or more aminoacid-N-carboxyanhydride monomers to the metallacycle for a time andunder conditions effective to form a polypeptide having an end groupderived from the alloc-amino acid amide.